Clusia pratensis drought study using constructed bacterial communities
Introduction
Methods
Packages
library(tidyverse)## Loading tidyverse: ggplot2
## Loading tidyverse: tibble
## Loading tidyverse: tidyr
## Loading tidyverse: readr
## Loading tidyverse: purrr
## Loading tidyverse: dplyr## Warning: package 'ggplot2' was built under R version 3.4.4## Warning: package 'tibble' was built under R version 3.4.4## Warning: package 'tidyr' was built under R version 3.4.4## Warning: package 'dplyr' was built under R version 3.4.4## Conflicts with tidy packages ----------------------------------------------## filter(): dplyr, stats
## lag(): dplyr, statslibrary(plotrix)## Warning: package 'plotrix' was built under R version 3.4.4library(car)##
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## extractBiomass Measurements
biomass <- read.csv("~/Documents/LabStats/Clusia/GrowthMeasurements.csv", header = TRUE, stringsAsFactors = TRUE, na.strings = "NA")
biomass <- biomass %>%
arrange(Condition, Treatment, SampleNumber)## Warning: package 'bindrcpp' was built under R version 3.4.4Controls
No difference between Control plants and plants that were inoculated with autoclaved bacteria (Control Mock). Confirms that the inoculation process itself does not account for changes between control plants and sample plants.
controls <- biomass %>%
filter(Treatment == "Control" | Treatment == "Control Mock")Soil water
soilwater.control <- aov(SoilWater ~ Condition * Treatment, data = controls)
summary(soilwater.control) # no difference between control and control mock ## Df Sum Sq Mean Sq F value Pr(>F)
## Condition 1 15410 15410 52.400 1.98e-06 ***
## Treatment 1 63 63 0.214 0.650
## Condition:Treatment 1 156 156 0.531 0.477
## Residuals 16 4705 294
## ---
## Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1Total Dry Mass
totaldrywt.control <- aov(Total_DryWt ~ Condition * Treatment, data = controls)
summary(totaldrywt.control) # no difference between control and control mock ## Df Sum Sq Mean Sq F value Pr(>F)
## Condition 1 3061 3060.8 9.789 0.00648 **
## Treatment 1 2 1.5 0.005 0.94548
## Condition:Treatment 1 70 70.4 0.225 0.64152
## Residuals 16 5003 312.7
## ---
## Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1Root biomass
rootbiomass.control <- aov(RootBiomass ~ Condition * Treatment, data = controls)
summary(rootbiomass.control) # no difference between control and control mock## Df Sum Sq Mean Sq F value Pr(>F)
## Condition 1 1860.3 1860.3 9.699 0.00668 **
## Treatment 1 60.7 60.7 0.316 0.58159
## Condition:Treatment 1 31.9 31.9 0.166 0.68876
## Residuals 16 3068.8 191.8
## ---
## Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1Root:Shoot
rootshoot.control <- aov(RootShoot ~ Condition * Treatment, data = controls)
summary(rootshoot.control) # slight interaction term, but negligible## Df Sum Sq Mean Sq F value Pr(>F)
## Condition 1 4.381 4.381 3.181 0.0935 .
## Treatment 1 3.621 3.621 2.629 0.1244
## Condition:Treatment 1 8.276 8.276 6.010 0.0261 *
## Residuals 16 22.031 1.377
## ---
## Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1RMF
rmf.control <- aov(RMF ~ Condition * Treatment, data = controls)
summary(rmf.control)## Df Sum Sq Mean Sq F value Pr(>F)
## Condition 1 0.00920 0.00920 1.636 0.2191
## Treatment 1 0.00588 0.00588 1.045 0.3220
## Condition:Treatment 1 0.03466 0.03466 6.161 0.0245 *
## Residuals 16 0.09000 0.00563
## ---
## Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1# removing control mock
biomass <- biomass %>%
filter(Treatment != "Control Mock")Summary Stats
biomass.sum <- biomass %>%
group_by(Condition) %>%
summarise_at(c("PotWt", "Leaf_FreshWt", "SoilWater", "Total_DryWt", "RootBiomass", "RootShoot", "RMF"), funs(mean, sd, std.error))
View(biomass.sum)
biomass.sum2 <- biomass %>%
group_by(Condition, Treatment) %>%
summarise_at(c("PotWt", "Leaf_FreshWt", "SoilWater", "Total_DryWt", "RootBiomass", "RootShoot", "RMF"), funs(mean, sd, std.error))
View(biomass.sum2)Boxplots
Total Biomass
mytheme <- theme_bw() + theme(panel.grid.major = element_blank(), panel.grid.minor = element_blank())
ggplot(data = biomass, aes(x = Treatment, y = Total_DryWt, fill = Condition)) + geom_boxplot() + mytheme + scale_fill_manual(values=c("indianred3", "cadetblue3"), breaks=c("Well-watered", "Drought"),labels=c("Well-watered", "Drought")) + theme(legend.position = c(0.1, 0.85)) + ylab("Biomass (g DW)") + xlab("Constructed Community") + scale_x_discrete(limits = c("Control", "Nonfunctional Isolates", "Drought Isolates", "Phosphorus Isolates", "All Isolates"))
Root biomass
ggplot(data = biomass, aes(x = Treatment, y = RootBiomass, fill = Condition)) + geom_boxplot() + mytheme + scale_fill_manual(values=c("indianred3", "cadetblue3"), breaks=c("Well-watered", "Drought"),labels=c("Well-watered", "Drought")) + theme(legend.position = c(0.1, 0.85)) + ylab("Root Biomass (g DW)") + xlab("Constructed Community") + scale_x_discrete(limits = c("Control", "Nonfunctional Isolates", "Drought Isolates", "Phosphorus Isolates", "All Isolates"))
Possible trend in both total biomass and root biomass is that both Drought and Phosphorus communities have consistently higher means than the control, nonfunctional isolates, and all isolates plants. Although not statistically significant, they suggest that there may be an advantage to the plants to have specifically these specialized constructed communities during drought conditions.
Root:Shoot
ggplot(data = biomass, aes(x = Treatment, y = RootShoot, fill = Condition)) + geom_boxplot() + mytheme + scale_fill_manual(values=c("indianred3", "cadetblue3"), breaks=c("Well-watered", "Drought"),labels=c("Well-watered", "Drought")) + theme(legend.position = c(0.1, 0.85)) + ylab("Root : Shoot") + xlab("Constructed Community") + scale_x_discrete(limits = c("Control", "Nonfunctional Isolates", "Drought Isolates", "Phosphorus Isolates", "All Isolates"))
Root Mass Fraction
ggplot(data = biomass, aes(x = Treatment, y = RMF, fill = Condition)) + geom_boxplot() + mytheme + scale_fill_manual(values=c("indianred3", "cadetblue3"), breaks=c("Well-watered", "Drought"),labels=c("Well-watered", "Drought")) + theme(legend.position = c(0.1, 0.85)) + ylab("Root Mass Fraction") + xlab("Constructed Community") + scale_x_discrete(limits = c("Control", "Nonfunctional Isolates", "Drought Isolates", "Phosphorus Isolates", "All Isolates"))
ANOVAs
Soil Water
soilwater.aov <- aov(SoilWater ~ Condition*Treatment, data = biomass)
summary(soilwater.aov) ## Df Sum Sq Mean Sq F value Pr(>F)
## Condition 1 55623 55623 222.891 <2e-16 ***
## Treatment 4 1150 287 1.152 0.341
## Condition:Treatment 4 1570 393 1.573 0.193
## Residuals 60 14973 250
## ---
## Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1# homogeneity of variance
plot(soilwater.aov, 1) 
leveneTest(SoilWater ~ Condition*Treatment, data = biomass) # testing normality
plot(soilwater.aov, 2)
water.residuals <- residuals(object = soilwater.aov)
shapiro.test(x = water.residuals) ##
## Shapiro-Wilk normality test
##
## data: water.residuals
## W = 0.83065, p-value = 1.721e-07ggplot(data = biomass, aes(x = Treatment, y = SoilWater)) + geom_boxplot() + facet_grid(. ~ Condition) + mytheme
Total Dry Mass
# density histogram
ggplot(data = biomass, aes(x = Total_DryWt)) + geom_histogram(aes(y=..density..), binwidth = 15, color="black", fill="gray") + geom_density(alpha = 0.2, fill = "#FF6666") + theme_classic() + xlab("Biomass (g DW)") + ylab("Density") + mytheme + ggtitle("Total Dry Mass Density Histogram")
# histogram by Condition (water treatment)
mu <- biomass %>%
group_by(Condition) %>%
summarise(mean = mean(Total_DryWt))
ggplot(data = biomass, aes(x = Total_DryWt, fill = Condition)) + geom_histogram(binwidth = 15, position = "identity", alpha = 0.5) + geom_vline(data = mu, aes(xintercept=mean, color=Condition), linetype="dashed") + scale_color_manual(values=c("indianred3", "cadetblue3")) + theme_classic() + xlab("Biomass (g DW)") + ylab("Count") + theme(legend.position = c(0.9, 0.9)) + ggtitle("Total Dry Mass Histogram")
totaldrywt.aov <- aov(Total_DryWt ~ Condition*Treatment, data = biomass)
summary(totaldrywt.aov) ## Df Sum Sq Mean Sq F value Pr(>F)
## Condition 1 18863 18863 32.100 4.41e-07 ***
## Treatment 4 1734 433 0.738 0.570
## Condition:Treatment 4 1757 439 0.747 0.564
## Residuals 60 35258 588
## ---
## Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1# homogeneity of variance
plot(totaldrywt.aov, 1)
leveneTest(Total_DryWt ~ Condition*Treatment, data = biomass) # testing normality
plot(totaldrywt.aov, 2)
totaldrywt.residuals <- residuals(object = totaldrywt.aov)
shapiro.test(x = totaldrywt.residuals) ##
## Shapiro-Wilk normality test
##
## data: totaldrywt.residuals
## W = 0.87475, p-value = 4.465e-06# identifying outliers
mod <- lm(Total_DryWt ~ Condition, data = biomass)
cooksd <- cooks.distance(mod)
plot(cooksd, pch="*", cex = 2, main = "Influential Obs in Total Dry Mass by Cooks Distance")
abline(h = 4*mean(cooksd, na.rm = T), col = "red")
text(x = 1: length(cooksd)+1, y = cooksd, labels = ifelse(cooksd>4*mean(cooksd, na.rm = T), names(cooksd), ""), col = "red")
# identifying rows with the outliers
biomass.out <- as.numeric(names(cooksd)[(cooksd > 4*mean(cooksd, na.rm = T))])
# removing outliers and testing impact
biomass <- biomass[-c(67,72), ]
ggqqplot(biomass$Total_DryWt)
shapiro.test(biomass$Total_DryWt) # completely normal##
## Shapiro-Wilk normality test
##
## data: biomass$Total_DryWt
## W = 0.84597, p-value = 5.813e-07# biomass total dry weight is now normal.
# rerunning biomass anova with removed outliers
totaldrywt.aov <- aov(Total_DryWt ~ Condition*Treatment, data = biomass)
summary(totaldrywt.aov) # same conclusion## Df Sum Sq Mean Sq F value Pr(>F)
## Condition 1 18731 18731 31.346 5.93e-07 ***
## Treatment 4 1761 440 0.737 0.571
## Condition:Treatment 4 1714 429 0.717 0.584
## Residuals 59 35256 598
## ---
## Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1Root Biomass
# density histogram
ggplot(data = biomass, aes(x = RootBiomass)) + geom_histogram(binwidth = 8, aes(y =..density..), colour = "black", fill = "white") + geom_density(alpha = 0.2, fill = "#FF6666") + mytheme + ggtitle("Root Biomass Density Diagram")
# by water treatment
mu2 <- biomass %>%
group_by(Condition) %>%
summarise(mean = mean(RootBiomass))
ggplot(data = biomass, aes(x = RootBiomass, fill = Condition)) + geom_histogram(binwidth =9, position = "identity", alpha = 0.5) + geom_vline(data = mu2, aes(xintercept=mean, color=Condition), linetype="dashed") + scale_color_manual(values=c("indianred3", "cadetblue3")) + theme_classic() + xlab("Root Biomass (g DW)") + ylab("Count") + theme(legend.position = c(0.9, 0.9)) + mytheme + ggtitle("Root Biomass Histogram")
# ANOVA
rootbiomass.aov <- aov(RootBiomass ~ Condition*Treatment, data = biomass)
summary(rootbiomass.aov) ## Df Sum Sq Mean Sq F value Pr(>F)
## Condition 1 9152 9152 24.003 7.85e-06 ***
## Treatment 4 1171 293 0.768 0.551
## Condition:Treatment 4 603 151 0.396 0.811
## Residuals 59 22497 381
## ---
## Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1# homogeneity of variance
plot(rootbiomass.aov, 1)
leveneTest(RootBiomass ~ Condition*Treatment, data = biomass) # normality test
plot(rootbiomass.aov, 2)
rootbiomass.residuals <- residuals(object = rootbiomass.aov)
shapiro.test(x = rootbiomass.residuals)##
## Shapiro-Wilk normality test
##
## data: rootbiomass.residuals
## W = 0.87195, p-value = 4.087e-06# identifying outliers
mod2 <- lm(RootBiomass ~ Condition, data = biomass)
cooksd2 <- cooks.distance(mod2)
plot(cooksd2, pch = "*", cex = 2, main = "Influential Observations in Root Biomass")
abline(h = 4*mean(cooksd2, na.rm = T), col = "red")
text(x=1: length(cooksd2) + 1, y= cooksd2, labels = ifelse(cooksd2>4*mean(cooksd2, na.rm = T), names(cooksd2), ""), col = "red")
# same samples are causing the irregularity
rootbiomass.out <- as.numeric(names(cooksd2)[(cooksd2 > 4*mean(cooksd2, na.rm = T))])
head(biomass[rootbiomass.out, ])# removing outliers and testing impact
biomass <- biomass[-c(67,72), ]
ggqqplot(biomass$RootBiomass)
shapiro.test(biomass$RootBiomass)##
## Shapiro-Wilk normality test
##
## data: biomass$RootBiomass
## W = 0.84494, p-value = 6.277e-07Root:Shoot
# density histogram
ggplot(data = biomass, aes(x = RootShoot)) + geom_histogram(binwidth = 0.8, aes(y =..density..), colour = "black", fill = "white") + geom_density(alpha = 0.2, fill = "#FF6666") + mytheme + ggtitle("Root:Shoot Density Histogram")
# by water treatment
mu3 <- biomass %>%
group_by(Condition) %>%
summarise(mean = mean(RootShoot))
ggplot(data = biomass, aes(x = RootShoot, fill = Condition)) + geom_histogram(binwidth = 0.8, position = "identity", alpha = 0.5) + geom_vline(data = mu3, aes(xintercept=mean, color=Condition), linetype="dashed") + scale_color_manual(values=c("indianred3", "cadetblue3")) + theme_classic() + xlab("RootShoot") + ylab("Count") + theme(legend.position = c(0.9, 0.9)) + mytheme + ggtitle("Root:Shoot Histogram")
# ANOVA
rootshoot.aov <- aov(RootShoot ~ Condition*Treatment, data = biomass)
summary(rootshoot.aov) ## Df Sum Sq Mean Sq F value Pr(>F)
## Condition 1 3.83 3.831 0.761 0.3865
## Treatment 4 50.40 12.601 2.504 0.0519 .
## Condition:Treatment 4 34.05 8.512 1.691 0.1643
## Residuals 58 291.87 5.032
## ---
## Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1# homogeneity of variance
plot(rootshoot.aov, 1) 
leveneTest(RootShoot ~ Condition*Treatment, data = biomass)# normality test
plot(rootshoot.aov, 2)
rootshoot.residuals <- residuals(object = rootshoot.aov)
shapiro.test(x = rootshoot.residuals) ##
## Shapiro-Wilk normality test
##
## data: rootshoot.residuals
## W = 0.92123, p-value = 0.0003675# identifying outliers
mod3 <- lm(RootShoot ~ Condition, data = biomass)
cooksd3 <- cooks.distance(mod3)
plot(cooksd3, pch = "*", cex = 2, main = "Influential Observations in RootShoot")
abline(h = 4*mean(cooksd3, na.rm = T), col = "red")
text(x=1: length(cooksd3) + 1, y= cooksd3, labels = ifelse(cooksd3>4*mean(cooksd3, na.rm = T), names(cooksd3), ""), col = "red")
rootbiomass.out <- as.numeric(names(cooksd3)[(cooksd3 > 4*mean(cooksd3, na.rm = T))])
head(biomass[rootbiomass.out, ])# removing outliers and testing impact
biomass.rootshoot <- biomass[-c(10,42, 51, 54), ]
ggqqplot(biomass.rootshoot$RootShoot)
shapiro.test(biomass.rootshoot$RootShoot)##
## Shapiro-Wilk normality test
##
## data: biomass.rootshoot$RootShoot
## W = 0.84147, p-value = 9.126e-07RMF
# density histogram
ggplot(data = biomass, aes(x = RMF)) + geom_histogram(binwidth = 0.03, aes(y =..density..), colour = "black", fill = "white") + geom_density(alpha = 0.2, fill = "#FF6666") + mytheme + ggtitle("Root Mass Fraction Density Histogram")
# by water treatment
mu4 <- biomass%>%
group_by(Condition) %>%
summarise(mean = mean(RMF))
ggplot(data = biomass, aes(x = RMF, fill = Condition)) + geom_histogram(binwidth = 0.03, position = "identity", alpha = 0.5) + geom_vline(data = mu4, aes(xintercept=mean, color=Condition), linetype="dashed") + scale_color_manual(values=c("indianred3", "cadetblue3")) + theme_classic() + xlab("Root Mass Fraction") + ylab("Count") + theme(legend.position = c(0.9, 0.9)) + mytheme + ggtitle("Root Mass Fraction Histogram")
# ANOVA
rmf.aov <- aov(RMF~ Condition*Treatment, data = biomass)
summary(rmf.aov) ## Df Sum Sq Mean Sq F value Pr(>F)
## Condition 1 0.0005 0.000502 0.058 0.8108
## Treatment 4 0.0871 0.021787 2.511 0.0514 .
## Condition:Treatment 4 0.0324 0.008109 0.935 0.4504
## Residuals 58 0.5032 0.008676
## ---
## Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1# homogeneity of variance
plot(rmf.aov, 1)
leveneTest(RMF ~ Condition*Treatment, data = biomass) # normality test
plot(rmf.aov, 2)
rmf.residuals <- residuals(object = rmf.aov)
shapiro.test(x = rmf.residuals) ##
## Shapiro-Wilk normality test
##
## data: rmf.residuals
## W = 0.97668, p-value = 0.2316Note: Root mass fraction = mass of fine root (g) / total biomass (g)
Physiology Measurements
Malic Acid Titrations
library(tidyverse)
clusia.malic <- read.csv("~/Documents/LabStats/Clusia/MalicAcid.csv", header = TRUE , sep = ",", stringsAsFactors = TRUE, na.strings = "na")Malic Acid Controls
# Week 0
wk0.controls <- clusia.malic %>%
filter(Treatment == "Control" | Treatment == "Control Mock") %>%
filter(Week == "Wk_0") %>%
arrange(Condition, Treatment)
malic.control0.aov <- aov(MalicAverage ~ Condition * Treatment, data = wk0.controls)
summary(malic.control0.aov) # not sigificant between autoclaved and inoculated controls## Df Sum Sq Mean Sq F value Pr(>F)
## Condition 1 1769 1769 0.409 0.546
## Treatment 1 2 2 0.000 0.983
## Condition:Treatment 1 43 43 0.010 0.923
## Residuals 6 25922 4320
## 1 observation deleted due to missingness# Week 2
wk2.controls <- clusia.malic %>%
filter(Treatment == "Control" | Treatment == "Control Mock") %>%
filter(Week == "Wk_2") %>%
arrange(Condition, Treatment)
malic.control2.aov <- aov(MalicAverage ~ Condition * Treatment, data = wk2.controls)
summary(malic.control2.aov) # not sigificant between autoclaved and inoculated controls## Df Sum Sq Mean Sq F value Pr(>F)
## Condition 1 150438 150438 11.524 0.00944 **
## Treatment 1 27151 27151 2.080 0.18724
## Condition:Treatment 1 1951 1951 0.149 0.70916
## Residuals 8 104439 13055
## ---
## Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1# Week 4
wk4.controls <- clusia.malic %>%
filter(Treatment == "Control" | Treatment == "Control Mock") %>%
filter(Week == "Wk_4") %>%
arrange(Condition, Treatment)
malic.control4.aov <- aov(MalicAverage ~ Condition * Treatment, data = wk4.controls)
summary(malic.control4.aov) # not sigificant between autoclaved and inoculated controls## Df Sum Sq Mean Sq F value Pr(>F)
## Condition 1 81042 81042 2.159 0.202
## Treatment 1 19763 19763 0.526 0.501
## Residuals 5 187700 37540# Week 6
wk6.controls <- clusia.malic %>%
filter(Treatment == "Control" | Treatment == "Control Mock") %>%
filter(Week == "Wk_6") %>%
arrange(Condition, Treatment)
malic.control6.aov <- aov(MalicAverage ~ Condition * Treatment, data = wk6.controls)
summary(malic.control6.aov) # not sigificant between autoclaved and inoculated controls## Df Sum Sq Mean Sq F value Pr(>F)
## Condition 1 4834 4834 0.713 0.426
## Treatment 1 1836 1836 0.271 0.619
## Condition:Treatment 1 4706 4706 0.695 0.432
## Residuals 7 47427 6775# Week 8
wk8.controls <- clusia.malic %>%
filter(Treatment == "Control" | Treatment == "Control Mock") %>%
filter(Week == "Wk_8") %>%
arrange(Condition, Treatment)
malic.control8.aov <- aov(MalicAverage ~ Condition * Treatment, data = wk8.controls)
summary(malic.control8.aov) # not sigificant between autoclaved and inoculated controls## Df Sum Sq Mean Sq F value Pr(>F)
## Condition 1 516094 516094 141.946 2.26e-06 ***
## Treatment 1 112 112 0.031 0.865
## Condition:Treatment 1 10 10 0.003 0.960
## Residuals 8 29087 3636
## ---
## Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1# removing Control Mock & fixing 'Null isolates'
clusia.malic <- clusia.malic %>%
filter(Treatment != "Control Mock")
clusia.malic$Treatment[clusia.malic$Treatment == "Null Isolates"] <- "Nonfunctional Isolates"
clusia.malic$Treatment[clusia.malic$Treatment == "Null_Isolates"] <- "Nonfunctional Isolates"
clusia.malic$Treatment[clusia.malic$Treatment == "All_Isolates"] <- "All Isolates"
clusia.malic$Treatment[clusia.malic$Treatment == "Drought_Isolates"] <- "Drought Isolates"
clusia.malic$Treatment[clusia.malic$Treatment == "Phosphorus_Isolates"] <- "Phosphorus Isolates"
# arranging by Week, Water, and Treatment
clusia.malic <- clusia.malic %>%
arrange(Week, Condition, Treatment)Mean, SD, Std.Error
malic.sum <- clusia.malic %>%
group_by(Week, Condition, Treatment) %>%
summarise(n = n(),
mean_malic = mean(MalicAverage, na.rm = TRUE),
sd_malic = sd(MalicAverage, na.rm = TRUE),
se = sd(MalicAverage, na.rm = TRUE)/sqrt(n()))
View(malic.sum)Malic Acid ANOVAs
Week 0
# subsetting Week 0
malic.wk0 <- clusia.malic %>%
filter(Week == "Wk_0")
# density histogram
ggplot(data = malic.wk0, aes(x = MalicAverage)) + geom_histogram(binwidth = 25, aes(y =..density..), colour = "black", fill = "white") + geom_density(alpha = 0.2, fill = "#FF6666") ## Warning: Removed 3 rows containing non-finite values (stat_bin).## Warning: Removed 3 rows containing non-finite values (stat_density).
# by water treatment
malic.wk0.mu <- malic.wk0%>%
group_by(Condition) %>%
summarise(mean = mean(MalicAverage, na.rm = TRUE))
ggplot(data = malic.wk0, aes(x = MalicAverage, fill = Condition)) + geom_histogram(binwidth = 25, position = "identity", alpha = 0.5) + geom_vline(data = malic.wk0.mu, aes(xintercept=mean, color=Condition), linetype="dashed") + scale_color_manual(values=c("indianred3", "cadetblue3")) + theme_classic() + xlab("Malic Acid") + ylab("Count") + theme(legend.position = c(0.9, 0.9))## Warning: Removed 3 rows containing non-finite values (stat_bin).
# ANOVA
malic.wk0.aov <- aov(MalicAverage ~ Condition * Treatment, data = malic.wk0)
summary(malic.wk0.aov) # not sigificant between treatment## Df Sum Sq Mean Sq F value Pr(>F)
## Condition 1 71 71 0.016 0.901
## Treatment 5 12528 2506 0.564 0.726
## Condition:Treatment 4 10351 2588 0.582 0.680
## Residuals 16 71089 4443
## 3 observations deleted due to missingness# testing homogeneity of variances
plot(malic.wk0.aov, 1)
leveneTest(MalicAverage ~ Condition*Treatment, data = malic.wk0) # p > 0.05 means no violation of homogeneity of varaince# normality test
plot(malic.wk0.aov, 2)## Warning: not plotting observations with leverage one:
## 20
malic.wk0.residuals <- residuals(object = malic.wk0.aov)
shapiro.test(x = malic.wk0.residuals) # p > 0.05 means normal data ##
## Shapiro-Wilk normality test
##
## data: malic.wk0.residuals
## W = 0.95026, p-value = 0.2175ggqqplot(malic.wk0$MalicAverage)## Warning: Removed 3 rows containing non-finite values (stat_qq).## Warning: Removed 3 rows containing non-finite values (stat_qq_line).
## Warning: Removed 3 rows containing non-finite values (stat_qq_line).
shapiro.test(malic.wk0$MalicAverage)##
## Shapiro-Wilk normality test
##
## data: malic.wk0$MalicAverage
## W = 0.97483, p-value = 0.732Week 2
malic.wk2 <- clusia.malic %>%
filter(Week == "Wk_2")
malic.wk2.aov <- aov(MalicAverage ~ Condition * Treatment, data = malic.wk2)
summary(malic.wk2.aov)## Df Sum Sq Mean Sq F value Pr(>F)
## Condition 1 287098 287098 36.643 8.02e-06 ***
## Treatment 4 34396 8599 1.098 0.38626
## Condition:Treatment 4 211782 52946 6.757 0.00147 **
## Residuals 19 148867 7835
## ---
## Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
## 1 observation deleted due to missingnessWeek 4
# subset for Week 4
malic.wk4 <- clusia.malic %>%
filter(Week == "Wk_4")
# density histogram
ggplot(data = malic.wk4, aes(x = MalicAverage)) + geom_histogram(binwidth = 50, aes(y =..density..), colour = "black", fill = "white") + geom_density(alpha = 0.2, fill = "#FF6666") 
# by water treatment
malic.wk4.mu <- malic.wk4%>%
group_by(Condition) %>%
summarise(mean = mean(MalicAverage, na.rm = TRUE))
ggplot(data = malic.wk4, aes(x = MalicAverage, fill = Condition)) + geom_histogram(binwidth = 50, position = "identity", alpha = 0.5) + geom_vline(data = malic.wk4.mu, aes(xintercept=mean, color=Condition), linetype="dashed") + scale_color_manual(values=c("indianred3", "cadetblue3")) + theme_classic() + xlab("Malic Acid") + ylab("Count") + theme(legend.position = c(0.9, 0.9))
# ANOVA
malic.wk4.aov <- aov(MalicAverage ~ Condition * Treatment, data = malic.wk4)
summary(malic.wk4.aov)## Df Sum Sq Mean Sq F value Pr(>F)
## Condition 1 802650 802650 12.430 0.00306 **
## Treatment 4 133019 33255 0.515 0.72590
## Condition:Treatment 4 65953 16488 0.255 0.90188
## Residuals 15 968573 64572
## ---
## Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1# testing homogeneity of variances
plot(malic.wk4.aov, 1)
leveneTest(MalicAverage ~ Condition*Treatment, data = malic.wk4) # p > 0.05 means no violation of homogeneity of varaince# normality test
plot(malic.wk4.aov, 2)## Warning: not plotting observations with leverage one:
## 6, 18
malic.wk4.residuals <- residuals(object = malic.wk4.aov)
shapiro.test(x = malic.wk4.residuals) # p > 0.05 means normal data##
## Shapiro-Wilk normality test
##
## data: malic.wk4.residuals
## W = 0.97125, p-value = 0.6768ggqqplot(malic.wk4$MalicAverage)
shapiro.test(malic.wk4$MalicAverage)##
## Shapiro-Wilk normality test
##
## data: malic.wk4$MalicAverage
## W = 0.96563, p-value = 0.5375Week 6
malic.wk6 <- clusia.malic %>%
filter(Week == "Wk_6")
malic.wk6.aov <- aov(MalicAverage ~ Condition * Treatment, data = malic.wk6)
summary(malic.wk6.aov) # sigificant drought effect## Df Sum Sq Mean Sq F value Pr(>F)
## Condition 1 152326 152326 17.672 0.000437 ***
## Treatment 4 73866 18467 2.142 0.113048
## Condition:Treatment 4 168945 42236 4.900 0.006424 **
## Residuals 20 172390 8619
## ---
## Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1Week 8
# subsetting for Week 8
malic.wk8 <- clusia.malic %>%
filter(Week == "Wk_8")
# density histogram
ggplot(data = malic.wk8, aes(x = MalicAverage)) + geom_histogram(binwidth = 80, aes(y =..density..), colour = "black", fill = "white") + geom_density(alpha = 0.2, fill = "#FF6666") 
# by water treatment
malic.wk8.mu <- malic.wk8%>%
group_by(Condition) %>%
summarise(mean = mean(MalicAverage, na.rm = TRUE))
ggplot(data = malic.wk8, aes(x = MalicAverage, fill = Condition)) + geom_histogram(binwidth = 80, position = "identity", alpha = 0.5) + geom_vline(data = malic.wk4.mu, aes(xintercept=mean, color=Condition), linetype="dashed") + scale_color_manual(values=c("indianred3", "cadetblue3")) + theme_classic() + xlab("Malic Acid") + ylab("Count") + theme(legend.position = c(0.9, 0.9))
# ANOVA
malic.wk8.aov <- aov(MalicAverage ~ Condition * Treatment, data = malic.wk8)
summary(malic.wk8.aov) # significant drought effect and microbial effect## Df Sum Sq Mean Sq F value Pr(>F)
## Condition 1 432864 432864 26.157 5.29e-05 ***
## Treatment 4 227707 56927 3.440 0.027 *
## Condition:Treatment 4 77288 19322 1.168 0.355
## Residuals 20 330968 16548
## ---
## Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1TukeyHSD(malic.wk8.aov)## Tukey multiple comparisons of means
## 95% family-wise confidence level
##
## Fit: aov(formula = MalicAverage ~ Condition * Treatment, data = malic.wk8)
##
## $Condition
## diff lwr upr p adj
## Well-watered-Drought 240.24 142.2563 338.2237 5.29e-05
##
## $Treatment
## diff lwr
## Control-All Isolates 134.41667 -87.828986
## Drought Isolates-All Isolates -10.90000 -233.145653
## Nonfunctional Isolates-All Isolates 218.45000 -3.795653
## Phosphorus Isolates-All Isolates 45.50000 -176.745653
## Drought Isolates-Control -145.31667 -367.562319
## Nonfunctional Isolates-Control 84.03333 -138.212319
## Phosphorus Isolates-Control -88.91667 -311.162319
## Nonfunctional Isolates-Drought Isolates 229.35000 7.104347
## Phosphorus Isolates-Drought Isolates 56.40000 -165.845653
## Phosphorus Isolates-Nonfunctional Isolates -172.95000 -395.195653
## upr p adj
## Control-All Isolates 356.66232 0.3955729
## Drought Isolates-All Isolates 211.34565 0.9998858
## Nonfunctional Isolates-All Isolates 440.69565 0.0554856
## Phosphorus Isolates-All Isolates 267.74565 0.9713947
## Drought Isolates-Control 76.92899 0.3217188
## Nonfunctional Isolates-Control 306.27899 0.7883022
## Phosphorus Isolates-Control 133.32899 0.7529027
## Nonfunctional Isolates-Drought Isolates 451.59565 0.0410511
## Phosphorus Isolates-Drought Isolates 278.64565 0.9392296
## Phosphorus Isolates-Nonfunctional Isolates 49.29565 0.1769778
##
## $`Condition:Treatment`
## diff
## Well-watered:All Isolates-Drought:All Isolates 176.266667
## Drought:Control-Drought:All Isolates 14.266667
## Well-watered:Control-Drought:All Isolates 430.833333
## Drought:Drought Isolates-Drought:All Isolates 4.433333
## Well-watered:Drought Isolates-Drought:All Isolates 150.033333
## Drought:Nonfunctional Isolates-Drought:All Isolates 160.833333
## Well-watered:Nonfunctional Isolates-Drought:All Isolates 452.333333
## Drought:Phosphorus Isolates-Drought:All Isolates 48.000000
## Well-watered:Phosphorus Isolates-Drought:All Isolates 219.266667
## Drought:Control-Well-watered:All Isolates -162.000000
## Well-watered:Control-Well-watered:All Isolates 254.566667
## Drought:Drought Isolates-Well-watered:All Isolates -171.833333
## Well-watered:Drought Isolates-Well-watered:All Isolates -26.233333
## Drought:Nonfunctional Isolates-Well-watered:All Isolates -15.433333
## Well-watered:Nonfunctional Isolates-Well-watered:All Isolates 276.066667
## Drought:Phosphorus Isolates-Well-watered:All Isolates -128.266667
## Well-watered:Phosphorus Isolates-Well-watered:All Isolates 43.000000
## Well-watered:Control-Drought:Control 416.566667
## Drought:Drought Isolates-Drought:Control -9.833333
## Well-watered:Drought Isolates-Drought:Control 135.766667
## Drought:Nonfunctional Isolates-Drought:Control 146.566667
## Well-watered:Nonfunctional Isolates-Drought:Control 438.066667
## Drought:Phosphorus Isolates-Drought:Control 33.733333
## Well-watered:Phosphorus Isolates-Drought:Control 205.000000
## Drought:Drought Isolates-Well-watered:Control -426.400000
## Well-watered:Drought Isolates-Well-watered:Control -280.800000
## Drought:Nonfunctional Isolates-Well-watered:Control -270.000000
## Well-watered:Nonfunctional Isolates-Well-watered:Control 21.500000
## Drought:Phosphorus Isolates-Well-watered:Control -382.833333
## Well-watered:Phosphorus Isolates-Well-watered:Control -211.566667
## Well-watered:Drought Isolates-Drought:Drought Isolates 145.600000
## Drought:Nonfunctional Isolates-Drought:Drought Isolates 156.400000
## Well-watered:Nonfunctional Isolates-Drought:Drought Isolates 447.900000
## Drought:Phosphorus Isolates-Drought:Drought Isolates 43.566667
## Well-watered:Phosphorus Isolates-Drought:Drought Isolates 214.833333
## Drought:Nonfunctional Isolates-Well-watered:Drought Isolates 10.800000
## Well-watered:Nonfunctional Isolates-Well-watered:Drought Isolates 302.300000
## Drought:Phosphorus Isolates-Well-watered:Drought Isolates -102.033333
## Well-watered:Phosphorus Isolates-Well-watered:Drought Isolates 69.233333
## Well-watered:Nonfunctional Isolates-Drought:Nonfunctional Isolates 291.500000
## Drought:Phosphorus Isolates-Drought:Nonfunctional Isolates -112.833333
## Well-watered:Phosphorus Isolates-Drought:Nonfunctional Isolates 58.433333
## Drought:Phosphorus Isolates-Well-watered:Nonfunctional Isolates -404.333333
## Well-watered:Phosphorus Isolates-Well-watered:Nonfunctional Isolates -233.066667
## Well-watered:Phosphorus Isolates-Drought:Phosphorus Isolates 171.266667
## lwr
## Well-watered:All Isolates-Drought:All Isolates -195.67212
## Drought:Control-Drought:All Isolates -357.67212
## Well-watered:Control-Drought:All Isolates 58.89455
## Drought:Drought Isolates-Drought:All Isolates -367.50545
## Well-watered:Drought Isolates-Drought:All Isolates -221.90545
## Drought:Nonfunctional Isolates-Drought:All Isolates -211.10545
## Well-watered:Nonfunctional Isolates-Drought:All Isolates 80.39455
## Drought:Phosphorus Isolates-Drought:All Isolates -323.93878
## Well-watered:Phosphorus Isolates-Drought:All Isolates -152.67212
## Drought:Control-Well-watered:All Isolates -533.93878
## Well-watered:Control-Well-watered:All Isolates -117.37212
## Drought:Drought Isolates-Well-watered:All Isolates -543.77212
## Well-watered:Drought Isolates-Well-watered:All Isolates -398.17212
## Drought:Nonfunctional Isolates-Well-watered:All Isolates -387.37212
## Well-watered:Nonfunctional Isolates-Well-watered:All Isolates -95.87212
## Drought:Phosphorus Isolates-Well-watered:All Isolates -500.20545
## Well-watered:Phosphorus Isolates-Well-watered:All Isolates -328.93878
## Well-watered:Control-Drought:Control 44.62788
## Drought:Drought Isolates-Drought:Control -381.77212
## Well-watered:Drought Isolates-Drought:Control -236.17212
## Drought:Nonfunctional Isolates-Drought:Control -225.37212
## Well-watered:Nonfunctional Isolates-Drought:Control 66.12788
## Drought:Phosphorus Isolates-Drought:Control -338.20545
## Well-watered:Phosphorus Isolates-Drought:Control -166.93878
## Drought:Drought Isolates-Well-watered:Control -798.33878
## Well-watered:Drought Isolates-Well-watered:Control -652.73878
## Drought:Nonfunctional Isolates-Well-watered:Control -641.93878
## Well-watered:Nonfunctional Isolates-Well-watered:Control -350.43878
## Drought:Phosphorus Isolates-Well-watered:Control -754.77212
## Well-watered:Phosphorus Isolates-Well-watered:Control -583.50545
## Well-watered:Drought Isolates-Drought:Drought Isolates -226.33878
## Drought:Nonfunctional Isolates-Drought:Drought Isolates -215.53878
## Well-watered:Nonfunctional Isolates-Drought:Drought Isolates 75.96122
## Drought:Phosphorus Isolates-Drought:Drought Isolates -328.37212
## Well-watered:Phosphorus Isolates-Drought:Drought Isolates -157.10545
## Drought:Nonfunctional Isolates-Well-watered:Drought Isolates -361.13878
## Well-watered:Nonfunctional Isolates-Well-watered:Drought Isolates -69.63878
## Drought:Phosphorus Isolates-Well-watered:Drought Isolates -473.97212
## Well-watered:Phosphorus Isolates-Well-watered:Drought Isolates -302.70545
## Well-watered:Nonfunctional Isolates-Drought:Nonfunctional Isolates -80.43878
## Drought:Phosphorus Isolates-Drought:Nonfunctional Isolates -484.77212
## Well-watered:Phosphorus Isolates-Drought:Nonfunctional Isolates -313.50545
## Drought:Phosphorus Isolates-Well-watered:Nonfunctional Isolates -776.27212
## Well-watered:Phosphorus Isolates-Well-watered:Nonfunctional Isolates -605.00545
## Well-watered:Phosphorus Isolates-Drought:Phosphorus Isolates -200.67212
## upr
## Well-watered:All Isolates-Drought:All Isolates 548.20545
## Drought:Control-Drought:All Isolates 386.20545
## Well-watered:Control-Drought:All Isolates 802.77212
## Drought:Drought Isolates-Drought:All Isolates 376.37212
## Well-watered:Drought Isolates-Drought:All Isolates 521.97212
## Drought:Nonfunctional Isolates-Drought:All Isolates 532.77212
## Well-watered:Nonfunctional Isolates-Drought:All Isolates 824.27212
## Drought:Phosphorus Isolates-Drought:All Isolates 419.93878
## Well-watered:Phosphorus Isolates-Drought:All Isolates 591.20545
## Drought:Control-Well-watered:All Isolates 209.93878
## Well-watered:Control-Well-watered:All Isolates 626.50545
## Drought:Drought Isolates-Well-watered:All Isolates 200.10545
## Well-watered:Drought Isolates-Well-watered:All Isolates 345.70545
## Drought:Nonfunctional Isolates-Well-watered:All Isolates 356.50545
## Well-watered:Nonfunctional Isolates-Well-watered:All Isolates 648.00545
## Drought:Phosphorus Isolates-Well-watered:All Isolates 243.67212
## Well-watered:Phosphorus Isolates-Well-watered:All Isolates 414.93878
## Well-watered:Control-Drought:Control 788.50545
## Drought:Drought Isolates-Drought:Control 362.10545
## Well-watered:Drought Isolates-Drought:Control 507.70545
## Drought:Nonfunctional Isolates-Drought:Control 518.50545
## Well-watered:Nonfunctional Isolates-Drought:Control 810.00545
## Drought:Phosphorus Isolates-Drought:Control 405.67212
## Well-watered:Phosphorus Isolates-Drought:Control 576.93878
## Drought:Drought Isolates-Well-watered:Control -54.46122
## Well-watered:Drought Isolates-Well-watered:Control 91.13878
## Drought:Nonfunctional Isolates-Well-watered:Control 101.93878
## Well-watered:Nonfunctional Isolates-Well-watered:Control 393.43878
## Drought:Phosphorus Isolates-Well-watered:Control -10.89455
## Well-watered:Phosphorus Isolates-Well-watered:Control 160.37212
## Well-watered:Drought Isolates-Drought:Drought Isolates 517.53878
## Drought:Nonfunctional Isolates-Drought:Drought Isolates 528.33878
## Well-watered:Nonfunctional Isolates-Drought:Drought Isolates 819.83878
## Drought:Phosphorus Isolates-Drought:Drought Isolates 415.50545
## Well-watered:Phosphorus Isolates-Drought:Drought Isolates 586.77212
## Drought:Nonfunctional Isolates-Well-watered:Drought Isolates 382.73878
## Well-watered:Nonfunctional Isolates-Well-watered:Drought Isolates 674.23878
## Drought:Phosphorus Isolates-Well-watered:Drought Isolates 269.90545
## Well-watered:Phosphorus Isolates-Well-watered:Drought Isolates 441.17212
## Well-watered:Nonfunctional Isolates-Drought:Nonfunctional Isolates 663.43878
## Drought:Phosphorus Isolates-Drought:Nonfunctional Isolates 259.10545
## Well-watered:Phosphorus Isolates-Drought:Nonfunctional Isolates 430.37212
## Drought:Phosphorus Isolates-Well-watered:Nonfunctional Isolates -32.39455
## Well-watered:Phosphorus Isolates-Well-watered:Nonfunctional Isolates 138.87212
## Well-watered:Phosphorus Isolates-Drought:Phosphorus Isolates 543.20545
## p adj
## Well-watered:All Isolates-Drought:All Isolates 0.7947843
## Drought:Control-Drought:All Isolates 1.0000000
## Well-watered:Control-Drought:All Isolates 0.0154590
## Drought:Drought Isolates-Drought:All Isolates 1.0000000
## Well-watered:Drought Isolates-Drought:All Isolates 0.9038592
## Drought:Nonfunctional Isolates-Drought:All Isolates 0.8641714
## Well-watered:Nonfunctional Isolates-Drought:All Isolates 0.0099410
## Drought:Phosphorus Isolates-Drought:All Isolates 0.9999755
## Well-watered:Phosphorus Isolates-Drought:All Isolates 0.5547819
## Drought:Control-Well-watered:All Isolates 0.8594242
## Well-watered:Control-Well-watered:All Isolates 0.3628413
## Drought:Drought Isolates-Well-watered:All Isolates 0.8161033
## Well-watered:Drought Isolates-Well-watered:All Isolates 0.9999999
## Drought:Nonfunctional Isolates-Well-watered:All Isolates 1.0000000
## Well-watered:Nonfunctional Isolates-Well-watered:All Isolates 0.2669553
## Drought:Phosphorus Isolates-Well-watered:All Isolates 0.9598867
## Well-watered:Phosphorus Isolates-Well-watered:All Isolates 0.9999904
## Well-watered:Control-Drought:Control 0.0206593
## Drought:Drought Isolates-Drought:Control 1.0000000
## Well-watered:Drought Isolates-Drought:Control 0.9441305
## Drought:Nonfunctional Isolates-Drought:Control 0.9149227
## Well-watered:Nonfunctional Isolates-Drought:Control 0.0133322
## Drought:Phosphorus Isolates-Drought:Control 0.9999988
## Well-watered:Phosphorus Isolates-Drought:Control 0.6379264
## Drought:Drought Isolates-Well-watered:Control 0.0169217
## Well-watered:Drought Isolates-Well-watered:Control 0.2484541
## Drought:Nonfunctional Isolates-Well-watered:Control 0.2920641
## Well-watered:Nonfunctional Isolates-Well-watered:Control 1.0000000
## Drought:Phosphorus Isolates-Well-watered:Control 0.0404567
## Well-watered:Phosphorus Isolates-Well-watered:Control 0.5996719
## Well-watered:Drought Isolates-Drought:Drought Isolates 0.9178606
## Drought:Nonfunctional Isolates-Drought:Drought Isolates 0.8814053
## Well-watered:Nonfunctional Isolates-Drought:Drought Isolates 0.0108926
## Drought:Phosphorus Isolates-Drought:Drought Isolates 0.9999893
## Well-watered:Phosphorus Isolates-Drought:Drought Isolates 0.5806005
## Drought:Nonfunctional Isolates-Well-watered:Drought Isolates 1.0000000
## Well-watered:Nonfunctional Isolates-Well-watered:Drought Isolates 0.1762492
## Drought:Phosphorus Isolates-Well-watered:Drought Isolates 0.9908546
## Well-watered:Phosphorus Isolates-Well-watered:Drought Isolates 0.9994979
## Well-watered:Nonfunctional Isolates-Drought:Nonfunctional Isolates 0.2101330
## Drought:Phosphorus Isolates-Drought:Nonfunctional Isolates 0.9820104
## Well-watered:Phosphorus Isolates-Drought:Nonfunctional Isolates 0.9998729
## Drought:Phosphorus Isolates-Well-watered:Nonfunctional Isolates 0.0264265
## Well-watered:Phosphorus Isolates-Well-watered:Nonfunctional Isolates 0.4759768
## Well-watered:Phosphorus Isolates-Drought:Phosphorus Isolates 0.8187525# testing homogeneity of variances
plot(malic.wk8.aov, 1)
leveneTest(MalicAverage ~ Condition*Treatment, data = malic.wk8) # p > 0.05 means no violation of homogeneity of varaince# normality test
plot(malic.wk8.aov, 2)
malic.wk8.residuals <- residuals(object = malic.wk8.aov)
shapiro.test(x = malic.wk8.residuals) # p > 0.05 means normal data##
## Shapiro-Wilk normality test
##
## data: malic.wk8.residuals
## W = 0.95804, p-value = 0.2757ggqqplot(malic.wk8$MalicAverage)
shapiro.test(malic.wk8$MalicAverage)##
## Shapiro-Wilk normality test
##
## data: malic.wk8$MalicAverage
## W = 0.92367, p-value = 0.03344# identifying outliers
wk8.mod <- lm(MalicAverage ~ Condition, data = malic.wk8)
cooksd.wk8 <- cooks.distance(wk8.mod)
plot(cooksd.wk8, pch = "*", cex = 2, main = "Influential Observations in Wk 8 Malic Acid")
abline(h = 4*mean(cooksd.wk8, na.rm = T), col = "red")
text(x=1: length(cooksd.wk8) + 1, y= cooksd.wk8, labels = ifelse(cooksd.wk8>4*mean(cooksd.wk8, na.rm = T), names(cooksd.wk8), ""), col = "red")
wk8.malic.out <- as.numeric(names(cooksd.wk8)[(cooksd.wk8 > 4*mean(cooksd.wk8, na.rm = T))])
head(biomass[wk8.malic.out, ])# removing outliers and testing impact
malic.wk8 <- malic.wk8[-c(24), ]
ggqqplot(malic.wk8$MalicAverage)
shapiro.test(malic.wk8$MalicAverage)##
## Shapiro-Wilk normality test
##
## data: malic.wk8$MalicAverage
## W = 0.92338, p-value = 0.03717clusia.boxplot <- ggplot(data = clusia.malic, aes(x = Treatment, y = MalicAverage, fill = Condition)) + geom_boxplot() + geom_point(aes(fill = Condition), size = 2, shape = 21, position = position_jitterdodge()) + facet_grid(. ~ Week)
clusia.boxplot <- clusia.boxplot + theme_bw() +scale_fill_manual(values=c("indianred3", "cadetblue3"), breaks=c("Well-watered", "Drought"),labels=c("Well-watered", "Drought")) + theme(panel.grid.major = element_blank(), panel.grid.minor = element_blank()) + theme(axis.title.x = element_blank()) + theme(axis.text.x = element_text(angle = 45, hjust = 1, vjust = 1)) + theme(legend.position = c(0.095, 0.9))
clusia.boxplot <- clusia.boxplot + ylab(expression(Malic~Acid~Concentration~(mu~mol~H~g^{-1})))
pdf("MalicAcid_Boxplot.pdf")
plot(clusia.boxplot)## Warning: Removed 4 rows containing non-finite values (stat_boxplot).## Warning: Removed 4 rows containing missing values (geom_point).dev.off()## quartz_off_screen
## 2At Week 0, both well-watered and drought plants show no difference in malic acid concentrations as expected. However, as the drought trial progresses, well-watered plants and drought plants begin to diverge significantly at Week 2 (p-value < 0.001). Higher amounts of malic acid in well-watered plants indicate higher CAM activity, which is in contrast with hypotheses that CAM activity would be highest in drought plants due to experiencing drought stress. Furthermore, there was no significant effect of microbial community until the last week (p- value < 0.05) with no interaction term, suggesting that availability of water is still the dominant driver of malic acid concentration. Tukeys HSD results indicate that although the differences in microbial communities are different, that these differences are occuring between well-watered microbial communities versus droughted microbial communities, verifying that the water availability is driving differences between plants and not the composition of the the microbial communities themselves.
Lipid Peroxidation Assay
clusia.lipid <- read.csv("~/Documents/Git/clusia_droughtexperiment/LipidPeroxidation.csv", header = TRUE , sep = ",", stringsAsFactors = TRUE, na.strings = "na")Lipid Peroxidation Controls
# Week 0
wk0.controls <- clusia.lipid %>%
filter(Treatment == "Control" | Treatment == "Control Mock") %>%
filter(Week == "Wk_0") %>%
arrange(Condition, Treatment)
lipid.control0.aov <- aov(LipidPerox ~ Condition * Treatment, data = wk0.controls)
summary(lipid.control0.aov) # not sigificant between autoclaved and inoculated controls## Df Sum Sq Mean Sq F value Pr(>F)
## Condition 1 1.0 1.05 0.019 0.895
## Treatment 1 20.1 20.07 0.357 0.569
## Condition:Treatment 1 44.0 44.03 0.782 0.406
## Residuals 7 393.9 56.27# Week 2
wk2.controls <- clusia.lipid %>%
filter(Treatment == "Control" | Treatment == "Control Mock") %>%
filter(Week == "Wk_2") %>%
arrange(Condition, Treatment)
lipid.control2.aov <- aov(LipidPerox ~ Condition * Treatment, data = wk2.controls)
summary(lipid.control2.aov) # not sigificant between autoclaved and inoculated controls## Df Sum Sq Mean Sq F value Pr(>F)
## Condition 1 14.1 14.07 0.274 0.615
## Treatment 1 42.4 42.42 0.826 0.390
## Condition:Treatment 1 41.9 41.95 0.817 0.393
## Residuals 8 410.9 51.36# Week 4
wk4.controls <- clusia.lipid %>%
filter(Treatment == "Control" | Treatment == "Control Mock") %>%
filter(Week == "Wk_4") %>%
arrange(Condition, Treatment)
lipid.control4.aov <- aov(LipidPerox ~ Condition * Treatment, data = wk4.controls)
summary(lipid.control4.aov) # not sigificant between autoclaved and inoculated controls## Df Sum Sq Mean Sq F value Pr(>F)
## Condition 1 30.3 30.30 0.167 0.695
## Treatment 1 19.8 19.76 0.109 0.751
## Condition:Treatment 1 68.7 68.65 0.378 0.558
## Residuals 7 1272.0 181.71# Week 6
wk6.controls <- clusia.lipid %>%
filter(Treatment == "Control" | Treatment == "Control Mock") %>%
filter(Week == "Wk_6") %>%
arrange(Condition, Treatment)
lipid.control6.aov <- aov(LipidPerox ~ Condition * Treatment, data = wk6.controls)
summary(lipid.control6.aov) # not sigificant between autoclaved and inoculated controls## Df Sum Sq Mean Sq F value Pr(>F)
## Condition 1 1362 1362.1 1.763 0.226
## Treatment 1 475 474.5 0.614 0.459
## Condition:Treatment 1 86 86.4 0.112 0.748
## Residuals 7 5409 772.6# Week 8
wk8.controls <- clusia.lipid %>%
filter(Treatment == "Control" | Treatment == "Control Mock") %>%
filter(Week == "Wk_8") %>%
arrange(Condition, Treatment)
lipid.control8.aov <- aov(LipidPerox ~ Condition * Treatment, data = wk8.controls)
summary(lipid.control8.aov) # not sigificant between autoclaved and inoculated controls## Df Sum Sq Mean Sq F value Pr(>F)
## Condition 1 1943.2 1943.2 9.942 0.0135 *
## Treatment 1 79.8 79.8 0.408 0.5408
## Condition:Treatment 1 5.9 5.9 0.030 0.8658
## Residuals 8 1563.6 195.5
## ---
## Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1# removing Control Mock
clusia.lipid <- clusia.lipid %>%
filter(Treatment != "Control Mock")
# arranging by Week, Water, and Treatment
clusia.lipid <- clusia.lipid %>%
arrange(Week, Condition, Treatment)Mean, SD, Std. Error
lipid.sum <- clusia.lipid %>%
group_by(Week, Condition, Treatment) %>%
summarise(n = n(),
mean_lipid = mean(LipidPerox, na.rm = TRUE),
sd_lipid = sd(LipidPerox, na.rm = TRUE),
se = sd(LipidPerox, na.rm = TRUE)/sqrt(n()))
View(lipid.sum)
lipid.errobars <- ggplot(data = lipid.sum, aes(x = Treatment, y = mean_lipid, fill = Condition)) + geom_bar(stat = "identity", color = "black", position = position_dodge(0.9)) + geom_errorbar(aes(ymin = mean_lipid-sd_lipid, ymax = mean_lipid+sd_lipid), width = 0.2, position = position_dodge(0.9)) + facet_grid(. ~ Week)
lipid.errobars <- lipid.errobars + theme_bw() + scale_fill_manual(values=c("indianred3", "cadetblue3"), breaks=c("Well-watered", "Drought"),labels=c("Well-watered", "Drought")) + theme(panel.grid.major = element_blank(), panel.grid.minor = element_blank()) + theme(axis.title.x = element_blank()) + theme(axis.text.x = element_text(angle = 45, hjust = 1, vjust = 1)) + theme(legend.position = c(0.1, 0.8)) + ylab(expression(Mean~Lipid~Peroxidation~(nmol~MDA~g^{-1})))
lipid.errobars## Warning: Removed 1 rows containing missing values (geom_errorbar).
Lipid Peroxidation ANOVAs
Week 0
# Week 0
lipid.wk0 <- clusia.lipid %>%
filter(Week == "Wk_0")
# density histogram
ggplot(data = lipid.wk0, aes(x = LipidPerox)) + geom_histogram(binwidth = 5, aes(y =..density..), colour = "black", fill = "white") + geom_density(alpha = 0.2, fill = "#FF6666") 
# by water treatment
lipid.wk0.mu <- lipid.wk0 %>%
group_by(Condition) %>%
summarise(mean = mean(LipidPerox, na.rm = TRUE))
ggplot(data = lipid.wk0, aes(x = LipidPerox, fill = Condition)) + geom_histogram(binwidth = 5, position = "identity", alpha = 0.5) + geom_vline(data = lipid.wk0.mu, aes(xintercept=mean, color=Condition), linetype="dashed") + scale_color_manual(values=c("indianred3", "cadetblue3")) + theme_classic() + xlab("Lipid Peroxidation") + ylab("Count") + theme(legend.position = c(0.9, 0.9))
# ANOVA
lipid.wk0.aov <- aov(LipidPerox ~ Condition * Treatment, data = lipid.wk0)
summary(lipid.wk0.aov) ## Df Sum Sq Mean Sq F value Pr(>F)
## Condition 1 15.8 15.81 0.173 0.682
## Treatment 4 285.4 71.35 0.781 0.551
## Condition:Treatment 4 222.9 55.72 0.610 0.660
## Residuals 20 1827.3 91.36# testing homogeneity of variances
plot(lipid.wk0.aov, 1)
leveneTest(LipidPerox ~ Condition*Treatment, data = lipid.wk0) # p > 0.05 means no violation of homogeneity of varaince# normality test
plot(lipid.wk0.aov, 2)
lipid.wk0.residuals <- residuals(object = lipid.wk0.aov)
shapiro.test(x = lipid.wk0.residuals) # p < 0.05 means normal data##
## Shapiro-Wilk normality test
##
## data: lipid.wk0.residuals
## W = 0.88192, p-value = 0.003121ggqqplot(lipid.wk0$LipidPerox)
shapiro.test(lipid.wk0$LipidPerox)##
## Shapiro-Wilk normality test
##
## data: lipid.wk0$LipidPerox
## W = 0.83772, p-value = 0.0003467# identifying outliers
lipid.wk0.mod <- lm(LipidPerox ~ Condition, data = lipid.wk0)
cooksd.wk0 <- cooks.distance(lipid.wk0.mod)
plot(cooksd.wk0, pch = "*", cex = 2, main = "Influential Observations in Wk 0 Lipid Peroxidation")
abline(h = 4*mean(cooksd.wk0, na.rm = T), col = "red")
text(x=1: length(cooksd.wk0) + 1, y= cooksd.wk0, labels = ifelse(cooksd.wk0>4*mean(cooksd.wk0, na.rm = T), names(cooksd.wk0), ""), col = "red")
wk0.lipid.out <- as.numeric(names(cooksd.wk0)[(cooksd.wk0 > 4*mean(cooksd.wk0, na.rm = T))])
head(clusia.lipid[wk0.lipid.out, ])# removing outliers and testing impact
lipid.wk0 <- lipid.wk0[-c(5), ]
ggqqplot(lipid.wk0$LipidPerox)
shapiro.test(lipid.wk0$LipidPerox)##
## Shapiro-Wilk normality test
##
## data: lipid.wk0$LipidPerox
## W = 0.82971, p-value = 0.000299Week 2
# Week 2
lipid.wk2 <- clusia.lipid %>%
filter(Week == "Wk_2")
lipid.wk2.aov <- aov(LipidPerox ~ Condition * Treatment, data = lipid.wk2)
summary(lipid.wk2.aov)## Df Sum Sq Mean Sq F value Pr(>F)
## Condition 1 38.6 38.64 1.297 0.269
## Treatment 4 205.3 51.33 1.723 0.186
## Condition:Treatment 4 126.6 31.65 1.062 0.402
## Residuals 19 566.0 29.79Week 4
# Week 4
lipid.wk4 <- clusia.lipid %>%
filter(Week == "Wk_4")
# density histogram
ggplot(data = lipid.wk4, aes(x = LipidPerox)) + geom_histogram(binwidth = 8, aes(y =..density..), colour = "black", fill = "white") + geom_density(alpha = 0.2, fill = "#FF6666") 
# by water treatment
lipid.wk4.mu <- lipid.wk4 %>%
group_by(Condition) %>%
summarise(mean = mean(LipidPerox, na.rm = TRUE))
ggplot(data = lipid.wk4, aes(x = LipidPerox, fill = Condition)) + geom_histogram(binwidth = 8, position = "identity", alpha = 0.5) + geom_vline(data = lipid.wk4.mu, aes(xintercept=mean, color=Condition), linetype="dashed") + scale_color_manual(values=c("indianred3", "cadetblue3")) + theme_classic() + xlab("Lipid Peroxidation") + ylab("Count") + theme(legend.position = c(0.9, 0.9))
# ANOVA
lipid.wk4.aov <- aov(LipidPerox ~ Condition * Treatment, data = lipid.wk4)
summary(lipid.wk4.aov) ## Df Sum Sq Mean Sq F value Pr(>F)
## Condition 1 11 11.4 0.054 0.820
## Treatment 4 1702 425.6 2.001 0.143
## Condition:Treatment 4 774 193.5 0.909 0.482
## Residuals 16 3404 212.7# testing homogeneity of variances
plot(lipid.wk4.aov, 1)
leveneTest(LipidPerox ~ Condition*Treatment, data = lipid.wk4) # normality test
plot(lipid.wk4.aov, 2)## Warning: not plotting observations with leverage one:
## 7
lipid.wk4.residuals <- residuals(object = lipid.wk4.aov)
shapiro.test(x = lipid.wk4.residuals) ##
## Shapiro-Wilk normality test
##
## data: lipid.wk4.residuals
## W = 0.96859, p-value = 0.5871ggqqplot(lipid.wk4$LipidPerox)
shapiro.test(lipid.wk4$LipidPerox)##
## Shapiro-Wilk normality test
##
## data: lipid.wk4$LipidPerox
## W = 0.96283, p-value = 0.4503Week 6
# Week 6
lipid.wk6 <- clusia.lipid %>%
filter(Week == "Wk_6")
lipid.wk6.aov <- aov(LipidPerox ~ Condition * Treatment, data = lipid.wk6)
summary(lipid.wk6.aov)## Df Sum Sq Mean Sq F value Pr(>F)
## Condition 1 3 2.9 0.009 0.9234
## Treatment 4 2790 697.5 2.261 0.0969 .
## Condition:Treatment 4 1515 378.9 1.228 0.3291
## Residuals 21 6479 308.5
## ---
## Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1Week 8
lipid.wk8 <- clusia.lipid %>%
filter(Week == "Wk_8")
# density histogram
ggplot(data = lipid.wk8, aes(x = LipidPerox)) + geom_histogram(binwidth = 10, aes(y =..density..), colour = "black", fill = "white") + geom_density(alpha = 0.2, fill = "#FF6666") 
# by water treatment
lipid.wk8.mu <- lipid.wk8 %>%
group_by(Condition) %>%
summarise(mean = mean(LipidPerox, na.rm = TRUE))
ggplot(data = lipid.wk8, aes(x = LipidPerox, fill = Condition)) + geom_histogram(binwidth = 10, position = "identity", alpha = 0.5) + geom_vline(data = lipid.wk8.mu, aes(xintercept=mean, color=Condition), linetype="dashed") + scale_color_manual(values=c("indianred3", "cadetblue3")) + theme_classic() + xlab("Lipid Peroxidation") + ylab("Count") + theme(legend.position = c(0.9, 0.9))
# ANOVA
lipid.wk8.aov <- aov(LipidPerox ~ Condition * Treatment, data = lipid.wk8)
summary(lipid.wk8.aov)## Df Sum Sq Mean Sq F value Pr(>F)
## Condition 1 1762 1762.2 7.625 0.01204 *
## Treatment 4 5627 1406.8 6.087 0.00226 **
## Condition:Treatment 4 475 118.7 0.513 0.72659
## Residuals 20 4622 231.1
## ---
## Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1TukeyHSD(lipid.wk8.aov)## Tukey multiple comparisons of means
## 95% family-wise confidence level
##
## Fit: aov(formula = LipidPerox ~ Condition * Treatment, data = lipid.wk8)
##
## $Condition
## diff lwr upr p adj
## Well-watered-Drought -15.32833 -26.90781 -3.748853 0.0120432
##
## $Treatment
## diff lwr upr
## Control-All Isolates -35.8098333 -62.07429 -9.545381
## Drought Isolates-All Isolates -24.8580000 -51.12245 1.406452
## Nonfunctional Isolates-All Isolates -34.7236667 -60.98812 -8.459214
## Phosphorus Isolates-All Isolates -35.3978333 -61.66229 -9.133381
## Drought Isolates-Control 10.9518333 -15.31262 37.216286
## Nonfunctional Isolates-Control 1.0861667 -25.17829 27.350619
## Phosphorus Isolates-Control 0.4120000 -25.85245 26.676452
## Nonfunctional Isolates-Drought Isolates -9.8656667 -36.13012 16.398786
## Phosphorus Isolates-Drought Isolates -10.5398333 -36.80429 15.724619
## Phosphorus Isolates-Nonfunctional Isolates -0.6741667 -26.93862 25.590286
## p adj
## Control-All Isolates 0.0047088
## Drought Isolates-All Isolates 0.0690768
## Nonfunctional Isolates-All Isolates 0.0062166
## Phosphorus Isolates-All Isolates 0.0052328
## Drought Isolates-Control 0.7244673
## Nonfunctional Isolates-Control 0.9999421
## Phosphorus Isolates-Control 0.9999988
## Nonfunctional Isolates-Drought Isolates 0.7921729
## Phosphorus Isolates-Drought Isolates 0.7508927
## Phosphorus Isolates-Nonfunctional Isolates 0.9999914
##
## $`Condition:Treatment`
## diff
## Well-watered:All Isolates-Drought:All Isolates -25.200333
## Drought:Control-Drought:All Isolates -36.388667
## Well-watered:Control-Drought:All Isolates -60.431333
## Drought:Drought Isolates-Drought:All Isolates -34.496667
## Well-watered:Drought Isolates-Drought:All Isolates -40.419667
## Drought:Nonfunctional Isolates-Drought:All Isolates -40.706667
## Well-watered:Nonfunctional Isolates-Drought:All Isolates -53.941000
## Drought:Phosphorus Isolates-Drought:All Isolates -43.877333
## Well-watered:Phosphorus Isolates-Drought:All Isolates -52.118667
## Drought:Control-Well-watered:All Isolates -11.188333
## Well-watered:Control-Well-watered:All Isolates -35.231000
## Drought:Drought Isolates-Well-watered:All Isolates -9.296333
## Well-watered:Drought Isolates-Well-watered:All Isolates -15.219333
## Drought:Nonfunctional Isolates-Well-watered:All Isolates -15.506333
## Well-watered:Nonfunctional Isolates-Well-watered:All Isolates -28.740667
## Drought:Phosphorus Isolates-Well-watered:All Isolates -18.677000
## Well-watered:Phosphorus Isolates-Well-watered:All Isolates -26.918333
## Well-watered:Control-Drought:Control -24.042667
## Drought:Drought Isolates-Drought:Control 1.892000
## Well-watered:Drought Isolates-Drought:Control -4.031000
## Drought:Nonfunctional Isolates-Drought:Control -4.318000
## Well-watered:Nonfunctional Isolates-Drought:Control -17.552333
## Drought:Phosphorus Isolates-Drought:Control -7.488667
## Well-watered:Phosphorus Isolates-Drought:Control -15.730000
## Drought:Drought Isolates-Well-watered:Control 25.934667
## Well-watered:Drought Isolates-Well-watered:Control 20.011667
## Drought:Nonfunctional Isolates-Well-watered:Control 19.724667
## Well-watered:Nonfunctional Isolates-Well-watered:Control 6.490333
## Drought:Phosphorus Isolates-Well-watered:Control 16.554000
## Well-watered:Phosphorus Isolates-Well-watered:Control 8.312667
## Well-watered:Drought Isolates-Drought:Drought Isolates -5.923000
## Drought:Nonfunctional Isolates-Drought:Drought Isolates -6.210000
## Well-watered:Nonfunctional Isolates-Drought:Drought Isolates -19.444333
## Drought:Phosphorus Isolates-Drought:Drought Isolates -9.380667
## Well-watered:Phosphorus Isolates-Drought:Drought Isolates -17.622000
## Drought:Nonfunctional Isolates-Well-watered:Drought Isolates -0.287000
## Well-watered:Nonfunctional Isolates-Well-watered:Drought Isolates -13.521333
## Drought:Phosphorus Isolates-Well-watered:Drought Isolates -3.457667
## Well-watered:Phosphorus Isolates-Well-watered:Drought Isolates -11.699000
## Well-watered:Nonfunctional Isolates-Drought:Nonfunctional Isolates -13.234333
## Drought:Phosphorus Isolates-Drought:Nonfunctional Isolates -3.170667
## Well-watered:Phosphorus Isolates-Drought:Nonfunctional Isolates -11.412000
## Drought:Phosphorus Isolates-Well-watered:Nonfunctional Isolates 10.063667
## Well-watered:Phosphorus Isolates-Well-watered:Nonfunctional Isolates 1.822333
## Well-watered:Phosphorus Isolates-Drought:Phosphorus Isolates -8.241333
## lwr
## Well-watered:All Isolates-Drought:All Isolates -69.15516
## Drought:Control-Drought:All Isolates -80.34349
## Well-watered:Control-Drought:All Isolates -104.38616
## Drought:Drought Isolates-Drought:All Isolates -78.45149
## Well-watered:Drought Isolates-Drought:All Isolates -84.37449
## Drought:Nonfunctional Isolates-Drought:All Isolates -84.66149
## Well-watered:Nonfunctional Isolates-Drought:All Isolates -97.89582
## Drought:Phosphorus Isolates-Drought:All Isolates -87.83216
## Well-watered:Phosphorus Isolates-Drought:All Isolates -96.07349
## Drought:Control-Well-watered:All Isolates -55.14316
## Well-watered:Control-Well-watered:All Isolates -79.18582
## Drought:Drought Isolates-Well-watered:All Isolates -53.25116
## Well-watered:Drought Isolates-Well-watered:All Isolates -59.17416
## Drought:Nonfunctional Isolates-Well-watered:All Isolates -59.46116
## Well-watered:Nonfunctional Isolates-Well-watered:All Isolates -72.69549
## Drought:Phosphorus Isolates-Well-watered:All Isolates -62.63182
## Well-watered:Phosphorus Isolates-Well-watered:All Isolates -70.87316
## Well-watered:Control-Drought:Control -67.99749
## Drought:Drought Isolates-Drought:Control -42.06282
## Well-watered:Drought Isolates-Drought:Control -47.98582
## Drought:Nonfunctional Isolates-Drought:Control -48.27282
## Well-watered:Nonfunctional Isolates-Drought:Control -61.50716
## Drought:Phosphorus Isolates-Drought:Control -51.44349
## Well-watered:Phosphorus Isolates-Drought:Control -59.68482
## Drought:Drought Isolates-Well-watered:Control -18.02016
## Well-watered:Drought Isolates-Well-watered:Control -23.94316
## Drought:Nonfunctional Isolates-Well-watered:Control -24.23016
## Well-watered:Nonfunctional Isolates-Well-watered:Control -37.46449
## Drought:Phosphorus Isolates-Well-watered:Control -27.40082
## Well-watered:Phosphorus Isolates-Well-watered:Control -35.64216
## Well-watered:Drought Isolates-Drought:Drought Isolates -49.87782
## Drought:Nonfunctional Isolates-Drought:Drought Isolates -50.16482
## Well-watered:Nonfunctional Isolates-Drought:Drought Isolates -63.39916
## Drought:Phosphorus Isolates-Drought:Drought Isolates -53.33549
## Well-watered:Phosphorus Isolates-Drought:Drought Isolates -61.57682
## Drought:Nonfunctional Isolates-Well-watered:Drought Isolates -44.24182
## Well-watered:Nonfunctional Isolates-Well-watered:Drought Isolates -57.47616
## Drought:Phosphorus Isolates-Well-watered:Drought Isolates -47.41249
## Well-watered:Phosphorus Isolates-Well-watered:Drought Isolates -55.65382
## Well-watered:Nonfunctional Isolates-Drought:Nonfunctional Isolates -57.18916
## Drought:Phosphorus Isolates-Drought:Nonfunctional Isolates -47.12549
## Well-watered:Phosphorus Isolates-Drought:Nonfunctional Isolates -55.36682
## Drought:Phosphorus Isolates-Well-watered:Nonfunctional Isolates -33.89116
## Well-watered:Phosphorus Isolates-Well-watered:Nonfunctional Isolates -42.13249
## Well-watered:Phosphorus Isolates-Drought:Phosphorus Isolates -52.19616
## upr
## Well-watered:All Isolates-Drought:All Isolates 18.7544901
## Drought:Control-Drought:All Isolates 7.5661568
## Well-watered:Control-Drought:All Isolates -16.4765099
## Drought:Drought Isolates-Drought:All Isolates 9.4581568
## Well-watered:Drought Isolates-Drought:All Isolates 3.5351568
## Drought:Nonfunctional Isolates-Drought:All Isolates 3.2481568
## Well-watered:Nonfunctional Isolates-Drought:All Isolates -9.9861766
## Drought:Phosphorus Isolates-Drought:All Isolates 0.0774901
## Well-watered:Phosphorus Isolates-Drought:All Isolates -8.1638432
## Drought:Control-Well-watered:All Isolates 32.7664901
## Well-watered:Control-Well-watered:All Isolates 8.7238234
## Drought:Drought Isolates-Well-watered:All Isolates 34.6584901
## Well-watered:Drought Isolates-Well-watered:All Isolates 28.7354901
## Drought:Nonfunctional Isolates-Well-watered:All Isolates 28.4484901
## Well-watered:Nonfunctional Isolates-Well-watered:All Isolates 15.2141568
## Drought:Phosphorus Isolates-Well-watered:All Isolates 25.2778234
## Well-watered:Phosphorus Isolates-Well-watered:All Isolates 17.0364901
## Well-watered:Control-Drought:Control 19.9121568
## Drought:Drought Isolates-Drought:Control 45.8468234
## Well-watered:Drought Isolates-Drought:Control 39.9238234
## Drought:Nonfunctional Isolates-Drought:Control 39.6368234
## Well-watered:Nonfunctional Isolates-Drought:Control 26.4024901
## Drought:Phosphorus Isolates-Drought:Control 36.4661568
## Well-watered:Phosphorus Isolates-Drought:Control 28.2248234
## Drought:Drought Isolates-Well-watered:Control 69.8894901
## Well-watered:Drought Isolates-Well-watered:Control 63.9664901
## Drought:Nonfunctional Isolates-Well-watered:Control 63.6794901
## Well-watered:Nonfunctional Isolates-Well-watered:Control 50.4451568
## Drought:Phosphorus Isolates-Well-watered:Control 60.5088234
## Well-watered:Phosphorus Isolates-Well-watered:Control 52.2674901
## Well-watered:Drought Isolates-Drought:Drought Isolates 38.0318234
## Drought:Nonfunctional Isolates-Drought:Drought Isolates 37.7448234
## Well-watered:Nonfunctional Isolates-Drought:Drought Isolates 24.5104901
## Drought:Phosphorus Isolates-Drought:Drought Isolates 34.5741568
## Well-watered:Phosphorus Isolates-Drought:Drought Isolates 26.3328234
## Drought:Nonfunctional Isolates-Well-watered:Drought Isolates 43.6678234
## Well-watered:Nonfunctional Isolates-Well-watered:Drought Isolates 30.4334901
## Drought:Phosphorus Isolates-Well-watered:Drought Isolates 40.4971568
## Well-watered:Phosphorus Isolates-Well-watered:Drought Isolates 32.2558234
## Well-watered:Nonfunctional Isolates-Drought:Nonfunctional Isolates 30.7204901
## Drought:Phosphorus Isolates-Drought:Nonfunctional Isolates 40.7841568
## Well-watered:Phosphorus Isolates-Drought:Nonfunctional Isolates 32.5428234
## Drought:Phosphorus Isolates-Well-watered:Nonfunctional Isolates 54.0184901
## Well-watered:Phosphorus Isolates-Well-watered:Nonfunctional Isolates 45.7771568
## Well-watered:Phosphorus Isolates-Drought:Phosphorus Isolates 35.7134901
## p adj
## Well-watered:All Isolates-Drought:All Isolates 0.5898939
## Drought:Control-Drought:All Isolates 0.1604508
## Well-watered:Control-Drought:All Isolates 0.0029134
## Drought:Drought Isolates-Drought:All Isolates 0.2087756
## Well-watered:Drought Isolates-Drought:All Isolates 0.0878770
## Drought:Nonfunctional Isolates-Drought:All Isolates 0.0840372
## Well-watered:Nonfunctional Isolates-Drought:All Isolates 0.0091325
## Drought:Phosphorus Isolates-Drought:All Isolates 0.0506365
## Well-watered:Phosphorus Isolates-Drought:All Isolates 0.0125483
## Drought:Control-Well-watered:All Isolates 0.9946005
## Well-watered:Control-Well-watered:All Isolates 0.1888093
## Drought:Drought Isolates-Well-watered:All Isolates 0.9986372
## Well-watered:Drought Isolates-Well-watered:All Isolates 0.9589139
## Drought:Nonfunctional Isolates-Well-watered:All Isolates 0.9541233
## Well-watered:Nonfunctional Isolates-Well-watered:All Isolates 0.4208204
## Drought:Phosphorus Isolates-Well-watered:All Isolates 0.8751733
## Well-watered:Phosphorus Isolates-Well-watered:All Isolates 0.5057846
## Well-watered:Control-Drought:Control 0.6469344
## Drought:Drought Isolates-Drought:Control 1.0000000
## Well-watered:Drought Isolates-Drought:Control 0.9999987
## Drought:Nonfunctional Isolates-Drought:Control 0.9999976
## Well-watered:Nonfunctional Isolates-Drought:Control 0.9087740
## Drought:Phosphorus Isolates-Drought:Control 0.9997535
## Well-watered:Phosphorus Isolates-Drought:Control 0.9501329
## Drought:Drought Isolates-Well-watered:Control 0.5536878
## Well-watered:Drought Isolates-Well-watered:Control 0.8276459
## Drought:Nonfunctional Isolates-Well-watered:Control 0.8385258
## Well-watered:Nonfunctional Isolates-Well-watered:Control 0.9999240
## Drought:Phosphorus Isolates-Well-watered:Control 0.9334189
## Well-watered:Phosphorus Isolates-Well-watered:Control 0.9994306
## Well-watered:Drought Isolates-Drought:Drought Isolates 0.9999647
## Drought:Nonfunctional Isolates-Drought:Drought Isolates 0.9999474
## Well-watered:Nonfunctional Isolates-Drought:Drought Isolates 0.8488137
## Drought:Phosphorus Isolates-Drought:Drought Isolates 0.9985395
## Well-watered:Phosphorus Isolates-Drought:Drought Isolates 0.9068716
## Drought:Nonfunctional Isolates-Well-watered:Drought Isolates 1.0000000
## Well-watered:Nonfunctional Isolates-Well-watered:Drought Isolates 0.9803061
## Drought:Phosphorus Isolates-Well-watered:Drought Isolates 0.9999997
## Well-watered:Phosphorus Isolates-Well-watered:Drought Isolates 0.9925906
## Well-watered:Nonfunctional Isolates-Drought:Nonfunctional Isolates 0.9828754
## Drought:Phosphorus Isolates-Drought:Nonfunctional Isolates 0.9999998
## Well-watered:Phosphorus Isolates-Drought:Nonfunctional Isolates 0.9937820
## Drought:Phosphorus Isolates-Well-watered:Nonfunctional Isolates 0.9975143
## Well-watered:Phosphorus Isolates-Well-watered:Nonfunctional Isolates 1.0000000
## Well-watered:Phosphorus Isolates-Drought:Phosphorus Isolates 0.9994682# testing homogeneity of variances
plot(lipid.wk8.aov, 1)
leveneTest(LipidPerox ~ Condition*Treatment, data = lipid.wk8) # p > 0.05 means no violation of homogeneity of varaince# normality test
plot(lipid.wk8.aov, 2)
lipid.wk8.residuals <- residuals(object = lipid.wk8.aov)
shapiro.test(x = lipid.wk8.residuals)##
## Shapiro-Wilk normality test
##
## data: lipid.wk8.residuals
## W = 0.94322, p-value = 0.1111lipid.boxplot <- ggplot(data = clusia.lipid, aes(x = Treatment, y = LipidPerox, fill = Condition)) + geom_boxplot() + geom_point(aes(fill = Condition), size = 2, shape = 21, position = position_jitterdodge()) + facet_grid(. ~ Week)
lipid.boxplot <- lipid.boxplot + theme_bw() +scale_fill_manual(values=c("indianred3", "cadetblue3"), breaks=c("Well-watered", "Drought"),labels=c("Well-watered", "Drought")) + theme(panel.grid.major = element_blank(), panel.grid.minor = element_blank()) + theme(axis.title.x = element_blank()) + theme(axis.text.x = element_text(angle = 45, hjust = 1, vjust = 1)) + theme(legend.position = c(0.1, 0.8)) + ylab(expression(Lipid~Peroxidation~(nmol~MDA~g^{-1})))
lipid.boxplot <- lipid.boxplot + theme_bw() +scale_fill_manual(values=c("indianred3", "cadetblue3"), breaks=c("Well-watered", "Drought"),labels=c("Well-watered", "Drought")) + theme(panel.grid.major = element_blank(), panel.grid.minor = element_blank()) + theme(axis.title.x = element_blank()) + theme(axis.text.x = element_text(angle = 45, hjust = 1, vjust = 1)) + theme(legend.position = c(0.1, 0.8))## Scale for 'fill' is already present. Adding another scale for 'fill',
## which will replace the existing scale.pdf("LipidPerox_Boxplot.pdf")
plot(lipid.boxplot)
dev.off()## quartz_off_screen
## 2Lipd peroxidation did not differ signifcantly between well-watered plants and drought plants until the final week of the experiment. Overall, plants inoculated with all beneficial isolates (All Isolates) differed from control plants (Control), plants that received autoclaved bacteria (Control Mock), and plants that received isolates with no measured function (Nonfunctional) (p-value < 0.05). The greatest differences in lipid peroxidation occured between well-watered plants and drought plants, indepent of microbial community. Although there is a significant microbial effect (p-value < 0.01), there is no interaction term suggesting that lipid peroxidation in drought conditions is not tied to microbial community composition despite the general differences between plants incolated with beneficial isolates and those of the control.
Proline Assay
clusia.proline <- read.csv("~/Documents/Git/clusia_droughtexperiment/Proline.csv", header = TRUE , sep = ",", stringsAsFactors = TRUE, na.strings = "na")Proline Controls
# Week 0
wk0.controls <- clusia.proline %>%
filter(Treatment == "Control" | Treatment == "Control Mock") %>%
filter(Week == "Wk_0") %>%
arrange(Condition, Treatment)
proline.control0.aov <- aov(Proline ~ Condition * Treatment, data = wk0.controls)
summary(proline.control0.aov) # not sigificant between autoclaved and inoculated controls## Df Sum Sq Mean Sq F value Pr(>F)
## Condition 1 3.96 3.960 0.221 0.653
## Treatment 1 20.46 20.458 1.141 0.321
## Condition:Treatment 1 7.39 7.386 0.412 0.541
## Residuals 7 125.53 17.933# Week 2
wk2.controls <- clusia.proline %>%
filter(Treatment == "Control" | Treatment == "Control Mock") %>%
filter(Week == "Wk_2") %>%
arrange(Condition, Treatment)
proline.control2.aov <- aov(Proline ~ Condition * Treatment, data = wk2.controls)
summary(proline.control2.aov) # not sigificant between autoclaved and inoculated controls## Df Sum Sq Mean Sq F value Pr(>F)
## Condition 1 161.52 161.52 6.444 0.0348 *
## Treatment 1 0.12 0.12 0.005 0.9456
## Condition:Treatment 1 2.88 2.88 0.115 0.7433
## Residuals 8 200.52 25.06
## ---
## Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1# Week 4
wk4.controls <- clusia.proline %>%
filter(Treatment == "Control" | Treatment == "Control Mock") %>%
filter(Week == "Wk_4") %>%
arrange(Condition, Treatment)
proline.control4.aov <- aov(Proline ~ Condition * Treatment, data = wk4.controls)
summary(proline.control4.aov) # significant interaction term + significant at < 0.1 for drought and microbial effect## Df Sum Sq Mean Sq F value Pr(>F)
## Condition 1 21.15 21.146 4.229 0.0855 .
## Treatment 1 23.07 23.074 4.614 0.0753 .
## Condition:Treatment 1 30.84 30.845 6.168 0.0476 *
## Residuals 6 30.00 5.001
## ---
## Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1# Week 6
wk6.controls <- clusia.proline %>%
filter(Treatment == "Control" | Treatment == "Control Mock") %>%
filter(Week == "Wk_6") %>%
arrange(Condition, Treatment)
proline.control6.aov <- aov(Proline ~ Condition * Treatment, data = wk6.controls)
summary(proline.control6.aov) # significant drought effect## Df Sum Sq Mean Sq F value Pr(>F)
## Condition 1 510.5 510.5 17.722 0.00399 **
## Treatment 1 4.6 4.6 0.159 0.70219
## Condition:Treatment 1 51.8 51.8 1.800 0.22164
## Residuals 7 201.6 28.8
## ---
## Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1# Week 8
wk8.controls <- clusia.proline %>%
filter(Treatment == "Control" | Treatment == "Control Mock") %>%
filter(Week == "Wk_8") %>%
arrange(Condition, Treatment)
proline.control8.aov <- aov(Proline ~ Condition * Treatment, data = wk8.controls)
summary(proline.control8.aov) # significant drought effect## Df Sum Sq Mean Sq F value Pr(>F)
## Condition 1 544.8 544.8 36.230 0.000317 ***
## Treatment 1 13.0 13.0 0.867 0.379007
## Condition:Treatment 1 17.3 17.3 1.152 0.314418
## Residuals 8 120.3 15.0
## ---
## Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1# removing Control Mock & fixing 'Null isolates'
clusia.lipid <- clusia.lipid %>%
filter(Treatment != "Control Mock")
# arranging by Week, Water, and Treatment
clusia.lipid <- clusia.lipid %>%
arrange(Week, Condition, Treatment)Mean, SD, Std. Error
proline.sum <- clusia.proline %>%
group_by(Week, Condition, Treatment) %>%
summarise(n = n(),
mean_proline = mean(Proline, na.rm = TRUE),
sd_proline = sd(Proline, na.rm = TRUE),
se = sd(Proline, na.rm = TRUE)/sqrt(n()))
View(proline.sum)
proline.errobars <- ggplot(data = proline.sum, aes(x = Treatment, y = mean_proline, fill = Condition)) + geom_bar(stat = "identity", color = "black", position = position_dodge(0.9)) + geom_errorbar(aes(ymin = mean_proline-sd_proline, ymax = mean_proline+sd_proline), width = 0.2, position = position_dodge(0.9)) + facet_grid(. ~ Week)
proline.errobars <- proline.errobars + theme_bw() + scale_fill_manual(values=c("indianred3", "cadetblue3"), breaks=c("Well-watered", "Drought"),labels=c("Well-watered", "Drought")) + theme(panel.grid.major = element_blank(), panel.grid.minor = element_blank()) + theme(axis.title.x = element_blank()) + theme(axis.text.x = element_text(angle = 45, hjust = 1, vjust = 1)) + theme(legend.position = c(0.09, 0.85)) + ylab(expression(Mean~Proline~Concentration(mu~moles~g^{-1})))
proline.errobars## Warning: Removed 1 rows containing missing values (geom_errorbar).
Proline ANOVAs
Week 0
# Week 0
proline.wk0 <- clusia.proline %>%
filter(Week == "Wk_0")
# density histogram
ggplot(data = proline.wk0, aes(x = Proline)) + geom_histogram(binwidth = 2, aes(y =..density..), colour = "black", fill = "white") + geom_density(alpha = 0.2, fill = "#FF6666") 
# by water treatment
proline.wk0.mu <- proline.wk0 %>%
group_by(Condition) %>%
summarise(mean = mean(Proline, na.rm = TRUE))
ggplot(data = proline.wk0, aes(x = Proline, fill = Condition)) + geom_histogram(binwidth = 2, position = "identity", alpha = 0.5) + geom_vline(data = proline.wk0.mu, aes(xintercept=mean, color=Condition), linetype="dashed") + scale_color_manual(values=c("indianred3", "cadetblue3")) + theme_classic() + xlab("Proline") + ylab("Count") + theme(legend.position = c(0.9, 0.9))
# ANOVA
proline.wk0.aov <- aov(Proline ~ Condition * Treatment, data = proline.wk0)
summary(proline.wk0.aov) ## Df Sum Sq Mean Sq F value Pr(>F)
## Condition 1 16.4 16.435 0.769 0.390
## Treatment 5 42.7 8.536 0.399 0.844
## Condition:Treatment 5 43.0 8.610 0.403 0.842
## Residuals 23 491.7 21.380# testing homogeneity of variances
plot(proline.wk0.aov, 1)
leveneTest(Proline ~ Condition*Treatment, data = proline.wk0) # normality test
plot(proline.wk0.aov, 2)
proline.wk0.residuals <- residuals(object = proline.wk0.aov)
shapiro.test(x = proline.wk0.residuals)##
## Shapiro-Wilk normality test
##
## data: proline.wk0.residuals
## W = 0.97952, p-value = 0.7435Week 2
# Week 2
proline.wk2 <- clusia.proline %>%
filter(Week == "Wk_2")
# ANOVA
proline.wk2.aov <- aov(Proline ~ Condition * Treatment, data = proline.wk2)
summary(proline.wk2.aov) # significant drought effect (<0.1) and interaction term (< 0.05)## Df Sum Sq Mean Sq F value Pr(>F)
## Condition 1 45.18 45.18 3.425 0.0771 .
## Treatment 5 56.29 11.26 0.854 0.5266
## Condition:Treatment 5 204.85 40.97 3.106 0.0275 *
## Residuals 23 303.37 13.19
## ---
## Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1Week 4
# Week 4
proline.wk4 <- clusia.proline %>%
filter(Week == "Wk_4")
# density histogram
ggplot(data = proline.wk4, aes(x = Proline)) + geom_histogram(binwidth = 2.5, aes(y =..density..), colour = "black", fill = "white") + geom_density(alpha = 0.2, fill = "#FF6666") 
# by water treatment
proline.wk4.mu <- proline.wk4 %>%
group_by(Condition) %>%
summarise(mean = mean(Proline, na.rm = TRUE))
ggplot(data = proline.wk4, aes(x = Proline, fill = Condition)) + geom_histogram(binwidth = 2.5, position = "identity", alpha = 0.5) + geom_vline(data = proline.wk4.mu, aes(xintercept=mean, color=Condition), linetype="dashed") + scale_color_manual(values=c("indianred3", "cadetblue3")) + theme_classic() + xlab("Proline") + ylab("Count") + theme(legend.position = c(0.9, 0.9))
# ANOVA
proline.wk4.aov <- aov(Proline ~ Condition * Treatment, data = proline.wk4)
summary(proline.wk4.aov) # significant drought effect## Df Sum Sq Mean Sq F value Pr(>F)
## Condition 1 133.5 133.48 6.883 0.0167 *
## Treatment 5 87.2 17.45 0.900 0.5014
## Condition:Treatment 5 61.2 12.24 0.631 0.6782
## Residuals 19 368.4 19.39
## ---
## Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1# testing homogeneity of variances
plot(proline.wk4.aov, 1)
leveneTest(Proline ~ Condition*Treatment, data = proline.wk4)# normality test
plot(proline.wk4.aov, 2)## Warning: not plotting observations with leverage one:
## 24
proline.wk4.residuals <- residuals(object = proline.wk4.aov)
shapiro.test(x = proline.wk4.residuals)##
## Shapiro-Wilk normality test
##
## data: proline.wk4.residuals
## W = 0.96447, p-value = 0.381Week 6
proline.wk6 <- clusia.proline %>%
filter(Week == "Wk_6")
proline.wk6.aov <- aov(Proline ~ Condition * Treatment, data = proline.wk6)
summary(proline.wk6.aov) # significant drought effect (<0.001), microbial (<0.1), interaction (<0.1)## Df Sum Sq Mean Sq F value Pr(>F)
## Condition 1 1240.6 1240.6 36.804 2.44e-06 ***
## Treatment 5 407.4 81.5 2.417 0.0642 .
## Condition:Treatment 5 371.5 74.3 2.204 0.0858 .
## Residuals 25 842.7 33.7
## ---
## Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1TukeyHSD(proline.wk6.aov)## Tukey multiple comparisons of means
## 95% family-wise confidence level
##
## Fit: aov(formula = Proline ~ Condition * Treatment, data = proline.wk6)
##
## $Condition
## diff lwr upr p adj
## Well-watered-Drought -11.5851 -15.51809 -7.652106 2.4e-06
##
## $Treatment
## diff lwr
## Control-All Isolates -4.8047972 -15.281487
## Control Mock-All Isolates -5.8835069 -15.837894
## Drought Isolates-All Isolates -3.0572995 -12.621165
## Nonfunctional Isolates-All Isolates -9.2510069 -19.205394
## Phosphorus Isolates-All Isolates -8.9371736 -18.891561
## Control Mock-Control -1.0787096 -11.913068
## Drought Isolates-Control 1.7474977 -8.729192
## Nonfunctional Isolates-Control -4.4462096 -15.280568
## Phosphorus Isolates-Control -4.1323763 -14.966735
## Drought Isolates-Control Mock 2.8262074 -7.128180
## Nonfunctional Isolates-Control Mock -3.3675000 -13.697656
## Phosphorus Isolates-Control Mock -3.0536667 -13.383822
## Nonfunctional Isolates-Drought Isolates -6.1937074 -16.148094
## Phosphorus Isolates-Drought Isolates -5.8798741 -15.834261
## Phosphorus Isolates-Nonfunctional Isolates 0.3138333 -10.016322
## upr p adj
## Control-All Isolates 5.6718928 0.7189041
## Control Mock-All Isolates 4.0708802 0.4708478
## Drought Isolates-All Isolates 6.5065662 0.9184005
## Nonfunctional Isolates-All Isolates 0.7033802 0.0793997
## Phosphorus Isolates-All Isolates 1.0172135 0.0968288
## Control Mock-Control 9.7556490 0.9995874
## Drought Isolates-Control 12.2241877 0.9951258
## Nonfunctional Isolates-Control 6.3881490 0.8007091
## Phosphorus Isolates-Control 6.7019823 0.8441368
## Drought Isolates-Control Mock 12.7805945 0.9490482
## Nonfunctional Isolates-Control Mock 6.9626557 0.9120373
## Phosphorus Isolates-Control Mock 7.2764890 0.9400229
## Nonfunctional Isolates-Drought Isolates 3.7606797 0.4153427
## Phosphorus Isolates-Drought Isolates 4.0745130 0.4715137
## Phosphorus Isolates-Nonfunctional Isolates 10.6439890 0.9999988
##
## $`Condition:Treatment`
## diff
## Well-watered:All Isolates-Drought:All Isolates -18.9151667
## Drought:Control-Drought:All Isolates -4.7186667
## Well-watered:Control-Drought:All Isolates -23.4283333
## Drought:Control Mock-Drought:All Isolates -10.9186667
## Well-watered:Control Mock-Drought:All Isolates -20.8106667
## Drought:Drought Isolates-Drought:All Isolates -11.5366667
## Well-watered:Drought Isolates-Drought:All Isolates -13.1100000
## Drought:Nonfunctional Isolates-Drought:All Isolates -11.5086667
## Well-watered:Nonfunctional Isolates-Drought:All Isolates -26.9556667
## Drought:Phosphorus Isolates-Drought:All Isolates -14.9636667
## Well-watered:Phosphorus Isolates-Drought:All Isolates -22.8730000
## Drought:Control-Well-watered:All Isolates 14.1965000
## Well-watered:Control-Well-watered:All Isolates -4.5131667
## Drought:Control Mock-Well-watered:All Isolates 7.9965000
## Well-watered:Control Mock-Well-watered:All Isolates -1.8955000
## Drought:Drought Isolates-Well-watered:All Isolates 7.3785000
## Well-watered:Drought Isolates-Well-watered:All Isolates 5.8051667
## Drought:Nonfunctional Isolates-Well-watered:All Isolates 7.4065000
## Well-watered:Nonfunctional Isolates-Well-watered:All Isolates -8.0405000
## Drought:Phosphorus Isolates-Well-watered:All Isolates 3.9515000
## Well-watered:Phosphorus Isolates-Well-watered:All Isolates -3.9578333
## Well-watered:Control-Drought:Control -18.7096667
## Drought:Control Mock-Drought:Control -6.2000000
## Well-watered:Control Mock-Drought:Control -16.0920000
## Drought:Drought Isolates-Drought:Control -6.8180000
## Well-watered:Drought Isolates-Drought:Control -8.3913333
## Drought:Nonfunctional Isolates-Drought:Control -6.7900000
## Well-watered:Nonfunctional Isolates-Drought:Control -22.2370000
## Drought:Phosphorus Isolates-Drought:Control -10.2450000
## Well-watered:Phosphorus Isolates-Drought:Control -18.1543333
## Drought:Control Mock-Well-watered:Control 12.5096667
## Well-watered:Control Mock-Well-watered:Control 2.6176667
## Drought:Drought Isolates-Well-watered:Control 11.8916667
## Well-watered:Drought Isolates-Well-watered:Control 10.3183333
## Drought:Nonfunctional Isolates-Well-watered:Control 11.9196667
## Well-watered:Nonfunctional Isolates-Well-watered:Control -3.5273333
## Drought:Phosphorus Isolates-Well-watered:Control 8.4646667
## Well-watered:Phosphorus Isolates-Well-watered:Control 0.5553333
## Well-watered:Control Mock-Drought:Control Mock -9.8920000
## Drought:Drought Isolates-Drought:Control Mock -0.6180000
## Well-watered:Drought Isolates-Drought:Control Mock -2.1913333
## Drought:Nonfunctional Isolates-Drought:Control Mock -0.5900000
## Well-watered:Nonfunctional Isolates-Drought:Control Mock -16.0370000
## Drought:Phosphorus Isolates-Drought:Control Mock -4.0450000
## Well-watered:Phosphorus Isolates-Drought:Control Mock -11.9543333
## Drought:Drought Isolates-Well-watered:Control Mock 9.2740000
## Well-watered:Drought Isolates-Well-watered:Control Mock 7.7006667
## Drought:Nonfunctional Isolates-Well-watered:Control Mock 9.3020000
## Well-watered:Nonfunctional Isolates-Well-watered:Control Mock -6.1450000
## Drought:Phosphorus Isolates-Well-watered:Control Mock 5.8470000
## Well-watered:Phosphorus Isolates-Well-watered:Control Mock -2.0623333
## Well-watered:Drought Isolates-Drought:Drought Isolates -1.5733333
## Drought:Nonfunctional Isolates-Drought:Drought Isolates 0.0280000
## Well-watered:Nonfunctional Isolates-Drought:Drought Isolates -15.4190000
## Drought:Phosphorus Isolates-Drought:Drought Isolates -3.4270000
## Well-watered:Phosphorus Isolates-Drought:Drought Isolates -11.3363333
## Drought:Nonfunctional Isolates-Well-watered:Drought Isolates 1.6013333
## Well-watered:Nonfunctional Isolates-Well-watered:Drought Isolates -13.8456667
## Drought:Phosphorus Isolates-Well-watered:Drought Isolates -1.8536667
## Well-watered:Phosphorus Isolates-Well-watered:Drought Isolates -9.7630000
## Well-watered:Nonfunctional Isolates-Drought:Nonfunctional Isolates -15.4470000
## Drought:Phosphorus Isolates-Drought:Nonfunctional Isolates -3.4550000
## Well-watered:Phosphorus Isolates-Drought:Nonfunctional Isolates -11.3643333
## Drought:Phosphorus Isolates-Well-watered:Nonfunctional Isolates 11.9920000
## Well-watered:Phosphorus Isolates-Well-watered:Nonfunctional Isolates 4.0826667
## Well-watered:Phosphorus Isolates-Drought:Phosphorus Isolates -7.9093333
## lwr
## Well-watered:All Isolates-Drought:All Isolates -34.841512
## Drought:Control-Drought:All Isolates -23.754291
## Well-watered:Control-Drought:All Isolates -40.454313
## Drought:Control Mock-Drought:All Isolates -27.944647
## Well-watered:Control Mock-Drought:All Isolates -37.836647
## Drought:Drought Isolates-Drought:All Isolates -27.463012
## Well-watered:Drought Isolates-Drought:All Isolates -30.135980
## Drought:Nonfunctional Isolates-Drought:All Isolates -28.534647
## Well-watered:Nonfunctional Isolates-Drought:All Isolates -43.981647
## Drought:Phosphorus Isolates-Drought:All Isolates -31.989647
## Well-watered:Phosphorus Isolates-Drought:All Isolates -39.898980
## Drought:Control-Well-watered:All Isolates -3.862279
## Well-watered:Control-Well-watered:All Isolates -20.439512
## Drought:Control Mock-Well-watered:All Isolates -7.929846
## Well-watered:Control Mock-Well-watered:All Isolates -17.821846
## Drought:Drought Isolates-Well-watered:All Isolates -7.366431
## Well-watered:Drought Isolates-Well-watered:All Isolates -10.121179
## Drought:Nonfunctional Isolates-Well-watered:All Isolates -8.519846
## Well-watered:Nonfunctional Isolates-Well-watered:All Isolates -23.966846
## Drought:Phosphorus Isolates-Well-watered:All Isolates -11.974846
## Well-watered:Phosphorus Isolates-Well-watered:All Isolates -19.884179
## Well-watered:Control-Drought:Control -37.745291
## Drought:Control Mock-Drought:Control -25.235624
## Well-watered:Control Mock-Drought:Control -35.127624
## Drought:Drought Isolates-Drought:Control -24.876779
## Well-watered:Drought Isolates-Drought:Control -27.426957
## Drought:Nonfunctional Isolates-Drought:Control -25.825624
## Well-watered:Nonfunctional Isolates-Drought:Control -41.272624
## Drought:Phosphorus Isolates-Drought:Control -29.280624
## Well-watered:Phosphorus Isolates-Drought:Control -37.189957
## Drought:Control Mock-Well-watered:Control -4.516313
## Well-watered:Control Mock-Well-watered:Control -14.408313
## Drought:Drought Isolates-Well-watered:Control -4.034679
## Well-watered:Drought Isolates-Well-watered:Control -6.707647
## Drought:Nonfunctional Isolates-Well-watered:Control -5.106313
## Well-watered:Nonfunctional Isolates-Well-watered:Control -20.553313
## Drought:Phosphorus Isolates-Well-watered:Control -8.561313
## Well-watered:Phosphorus Isolates-Well-watered:Control -16.470647
## Well-watered:Control Mock-Drought:Control Mock -26.917980
## Drought:Drought Isolates-Drought:Control Mock -16.544346
## Well-watered:Drought Isolates-Drought:Control Mock -19.217313
## Drought:Nonfunctional Isolates-Drought:Control Mock -17.615980
## Well-watered:Nonfunctional Isolates-Drought:Control Mock -33.062980
## Drought:Phosphorus Isolates-Drought:Control Mock -21.070980
## Well-watered:Phosphorus Isolates-Drought:Control Mock -28.980313
## Drought:Drought Isolates-Well-watered:Control Mock -6.652346
## Well-watered:Drought Isolates-Well-watered:Control Mock -9.325313
## Drought:Nonfunctional Isolates-Well-watered:Control Mock -7.723980
## Well-watered:Nonfunctional Isolates-Well-watered:Control Mock -23.170980
## Drought:Phosphorus Isolates-Well-watered:Control Mock -11.178980
## Well-watered:Phosphorus Isolates-Well-watered:Control Mock -19.088313
## Well-watered:Drought Isolates-Drought:Drought Isolates -17.499679
## Drought:Nonfunctional Isolates-Drought:Drought Isolates -15.898346
## Well-watered:Nonfunctional Isolates-Drought:Drought Isolates -31.345346
## Drought:Phosphorus Isolates-Drought:Drought Isolates -19.353346
## Well-watered:Phosphorus Isolates-Drought:Drought Isolates -27.262679
## Drought:Nonfunctional Isolates-Well-watered:Drought Isolates -15.424647
## Well-watered:Nonfunctional Isolates-Well-watered:Drought Isolates -30.871647
## Drought:Phosphorus Isolates-Well-watered:Drought Isolates -18.879647
## Well-watered:Phosphorus Isolates-Well-watered:Drought Isolates -26.788980
## Well-watered:Nonfunctional Isolates-Drought:Nonfunctional Isolates -32.472980
## Drought:Phosphorus Isolates-Drought:Nonfunctional Isolates -20.480980
## Well-watered:Phosphorus Isolates-Drought:Nonfunctional Isolates -28.390313
## Drought:Phosphorus Isolates-Well-watered:Nonfunctional Isolates -5.033980
## Well-watered:Phosphorus Isolates-Well-watered:Nonfunctional Isolates -12.943313
## Well-watered:Phosphorus Isolates-Drought:Phosphorus Isolates -24.935313
## upr
## Well-watered:All Isolates-Drought:All Isolates -2.9888208
## Drought:Control-Drought:All Isolates 14.3169575
## Well-watered:Control-Drought:All Isolates -6.4023535
## Drought:Control Mock-Drought:All Isolates 6.1073132
## Well-watered:Control Mock-Drought:All Isolates -3.7846868
## Drought:Drought Isolates-Drought:All Isolates 4.3896792
## Well-watered:Drought Isolates-Drought:All Isolates 3.9159799
## Drought:Nonfunctional Isolates-Drought:All Isolates 5.5173132
## Well-watered:Nonfunctional Isolates-Drought:All Isolates -9.9296868
## Drought:Phosphorus Isolates-Drought:All Isolates 2.0623132
## Well-watered:Phosphorus Isolates-Drought:All Isolates -5.8470201
## Drought:Control-Well-watered:All Isolates 32.2552787
## Well-watered:Control-Well-watered:All Isolates 11.4131792
## Drought:Control Mock-Well-watered:All Isolates 23.9228458
## Well-watered:Control Mock-Well-watered:All Isolates 14.0308458
## Drought:Drought Isolates-Well-watered:All Isolates 22.1234311
## Well-watered:Drought Isolates-Well-watered:All Isolates 21.7315125
## Drought:Nonfunctional Isolates-Well-watered:All Isolates 23.3328458
## Well-watered:Nonfunctional Isolates-Well-watered:All Isolates 7.8858458
## Drought:Phosphorus Isolates-Well-watered:All Isolates 19.8778458
## Well-watered:Phosphorus Isolates-Well-watered:All Isolates 11.9685125
## Well-watered:Control-Drought:Control 0.3259575
## Drought:Control Mock-Drought:Control 12.8356242
## Well-watered:Control Mock-Drought:Control 2.9436242
## Drought:Drought Isolates-Drought:Control 11.2407787
## Well-watered:Drought Isolates-Drought:Control 10.6442908
## Drought:Nonfunctional Isolates-Drought:Control 12.2456242
## Well-watered:Nonfunctional Isolates-Drought:Control -3.2013758
## Drought:Phosphorus Isolates-Drought:Control 8.7906242
## Well-watered:Phosphorus Isolates-Drought:Control 0.8812908
## Drought:Control Mock-Well-watered:Control 29.5356465
## Well-watered:Control Mock-Well-watered:Control 19.6436465
## Drought:Drought Isolates-Well-watered:Control 27.8180125
## Well-watered:Drought Isolates-Well-watered:Control 27.3443132
## Drought:Nonfunctional Isolates-Well-watered:Control 28.9456465
## Well-watered:Nonfunctional Isolates-Well-watered:Control 13.4986465
## Drought:Phosphorus Isolates-Well-watered:Control 25.4906465
## Well-watered:Phosphorus Isolates-Well-watered:Control 17.5813132
## Well-watered:Control Mock-Drought:Control Mock 7.1339799
## Drought:Drought Isolates-Drought:Control Mock 15.3083458
## Well-watered:Drought Isolates-Drought:Control Mock 14.8346465
## Drought:Nonfunctional Isolates-Drought:Control Mock 16.4359799
## Well-watered:Nonfunctional Isolates-Drought:Control Mock 0.9889799
## Drought:Phosphorus Isolates-Drought:Control Mock 12.9809799
## Well-watered:Phosphorus Isolates-Drought:Control Mock 5.0716465
## Drought:Drought Isolates-Well-watered:Control Mock 25.2003458
## Well-watered:Drought Isolates-Well-watered:Control Mock 24.7266465
## Drought:Nonfunctional Isolates-Well-watered:Control Mock 26.3279799
## Well-watered:Nonfunctional Isolates-Well-watered:Control Mock 10.8809799
## Drought:Phosphorus Isolates-Well-watered:Control Mock 22.8729799
## Well-watered:Phosphorus Isolates-Well-watered:Control Mock 14.9636465
## Well-watered:Drought Isolates-Drought:Drought Isolates 14.3530125
## Drought:Nonfunctional Isolates-Drought:Drought Isolates 15.9543458
## Well-watered:Nonfunctional Isolates-Drought:Drought Isolates 0.5073458
## Drought:Phosphorus Isolates-Drought:Drought Isolates 12.4993458
## Well-watered:Phosphorus Isolates-Drought:Drought Isolates 4.5900125
## Drought:Nonfunctional Isolates-Well-watered:Drought Isolates 18.6273132
## Well-watered:Nonfunctional Isolates-Well-watered:Drought Isolates 3.1803132
## Drought:Phosphorus Isolates-Well-watered:Drought Isolates 15.1723132
## Well-watered:Phosphorus Isolates-Well-watered:Drought Isolates 7.2629799
## Well-watered:Nonfunctional Isolates-Drought:Nonfunctional Isolates 1.5789799
## Drought:Phosphorus Isolates-Drought:Nonfunctional Isolates 13.5709799
## Well-watered:Phosphorus Isolates-Drought:Nonfunctional Isolates 5.6616465
## Drought:Phosphorus Isolates-Well-watered:Nonfunctional Isolates 29.0179799
## Well-watered:Phosphorus Isolates-Well-watered:Nonfunctional Isolates 21.1086465
## Well-watered:Phosphorus Isolates-Drought:Phosphorus Isolates 9.1166465
## p adj
## Well-watered:All Isolates-Drought:All Isolates 0.0106562
## Drought:Control-Drought:All Isolates 0.9986294
## Well-watered:Control-Drought:All Isolates 0.0020620
## Drought:Control Mock-Drought:All Isolates 0.4992419
## Well-watered:Control Mock-Drought:All Isolates 0.0079152
## Drought:Drought Isolates-Drought:All Isolates 0.3294478
## Well-watered:Drought Isolates-Drought:All Isolates 0.2529134
## Drought:Nonfunctional Isolates-Drought:All Isolates 0.4244338
## Well-watered:Nonfunctional Isolates-Drought:All Isolates 0.0003285
## Drought:Phosphorus Isolates-Drought:All Isolates 0.1236067
## Well-watered:Phosphorus Isolates-Drought:All Isolates 0.0027496
## Drought:Control-Well-watered:All Isolates 0.2290954
## Well-watered:Control-Well-watered:All Isolates 0.9956679
## Drought:Control Mock-Well-watered:All Isolates 0.8017581
## Well-watered:Control Mock-Well-watered:All Isolates 0.9999990
## Drought:Drought Isolates-Well-watered:All Isolates 0.8048958
## Well-watered:Drought Isolates-Well-watered:All Isolates 0.9700448
## Drought:Nonfunctional Isolates-Well-watered:All Isolates 0.8651719
## Well-watered:Nonfunctional Isolates-Well-watered:All Isolates 0.7965601
## Drought:Phosphorus Isolates-Well-watered:All Isolates 0.9986183
## Well-watered:Phosphorus Isolates-Well-watered:All Isolates 0.9985986
## Well-watered:Control-Drought:Control 0.0571389
## Drought:Control Mock-Drought:Control 0.9868404
## Well-watered:Control Mock-Drought:Control 0.1558471
## Drought:Drought Isolates-Drought:Control 0.9618607
## Well-watered:Drought Isolates-Drought:Control 0.8996163
## Drought:Nonfunctional Isolates-Drought:Control 0.9742791
## Well-watered:Nonfunctional Isolates-Drought:Control 0.0125815
## Drought:Phosphorus Isolates-Drought:Control 0.7296076
## Well-watered:Phosphorus Isolates-Drought:Control 0.0714522
## Drought:Control Mock-Well-watered:Control 0.3108945
## Well-watered:Control Mock-Well-watered:Control 0.9999858
## Drought:Drought Isolates-Well-watered:Control 0.2903827
## Well-watered:Drought Isolates-Well-watered:Control 0.5787270
## Drought:Nonfunctional Isolates-Well-watered:Control 0.3754711
## Well-watered:Nonfunctional Isolates-Well-watered:Control 0.9997356
## Drought:Phosphorus Isolates-Well-watered:Control 0.8108849
## Well-watered:Phosphorus Isolates-Well-watered:Control 1.0000000
## Well-watered:Control Mock-Drought:Control Mock 0.6355962
## Drought:Drought Isolates-Drought:Control Mock 1.0000000
## Well-watered:Drought Isolates-Drought:Control Mock 0.9999977
## Drought:Nonfunctional Isolates-Drought:Control Mock 1.0000000
## Well-watered:Nonfunctional Isolates-Drought:Control Mock 0.0780933
## Drought:Phosphorus Isolates-Drought:Control Mock 0.9990626
## Well-watered:Phosphorus Isolates-Drought:Control Mock 0.3714821
## Drought:Drought Isolates-Well-watered:Control Mock 0.6326336
## Well-watered:Drought Isolates-Well-watered:Control Mock 0.8840431
## Drought:Nonfunctional Isolates-Well-watered:Control Mock 0.7122677
## Well-watered:Nonfunctional Isolates-Well-watered:Control Mock 0.9720479
## Drought:Phosphorus Isolates-Well-watered:Control Mock 0.9804445
## Well-watered:Phosphorus Isolates-Well-watered:Control Mock 0.9999988
## Well-watered:Drought Isolates-Drought:Drought Isolates 0.9999999
## Drought:Nonfunctional Isolates-Drought:Drought Isolates 1.0000000
## Well-watered:Nonfunctional Isolates-Drought:Drought Isolates 0.0640073
## Drought:Phosphorus Isolates-Drought:Drought Isolates 0.9996225
## Well-watered:Phosphorus Isolates-Drought:Drought Isolates 0.3528499
## Drought:Nonfunctional Isolates-Well-watered:Drought Isolates 0.9999999
## Well-watered:Nonfunctional Isolates-Well-watered:Drought Isolates 0.1928909
## Drought:Phosphorus Isolates-Well-watered:Drought Isolates 0.9999996
## Well-watered:Phosphorus Isolates-Well-watered:Drought Isolates 0.6526540
## Well-watered:Nonfunctional Isolates-Drought:Nonfunctional Isolates 0.1008700
## Drought:Phosphorus Isolates-Drought:Nonfunctional Isolates 0.9997828
## Well-watered:Phosphorus Isolates-Drought:Nonfunctional Isolates 0.4422979
## Drought:Phosphorus Isolates-Well-watered:Nonfunctional Isolates 0.3671742
## Well-watered:Phosphorus Isolates-Well-watered:Nonfunctional Isolates 0.9989810
## Well-watered:Phosphorus Isolates-Drought:Phosphorus Isolates 0.8659447Week 8
# Week 8
proline.wk8 <- clusia.proline %>%
filter(Week == "Wk_8")
# density histogram
ggplot(data = proline.wk8, aes(x = Proline)) + geom_histogram(binwidth = 4.5, aes(y =..density..), colour = "black", fill = "white") + geom_density(alpha = 0.2, fill = "#FF6666") 
# by water treatment
proline.wk8.mu <- proline.wk8 %>%
group_by(Condition) %>%
summarise(mean = mean(Proline, na.rm = TRUE))
ggplot(data = proline.wk8, aes(x = Proline, fill = Condition)) + geom_histogram(binwidth = 4.5, position = "identity", alpha = 0.5) + geom_vline(data = proline.wk8.mu, aes(xintercept=mean, color=Condition), linetype="dashed") + scale_color_manual(values=c("indianred3", "cadetblue3")) + theme_classic() + xlab("Proline") + ylab("Count") + theme(legend.position = c(0.9, 0.9))
# ANOVA
proline.wk8.aov <- aov(Proline ~ Condition * Treatment, data = proline.wk8)
summary(proline.wk8.aov) # significant drought effect (<0.001) and microbial effect (<0.1)## Df Sum Sq Mean Sq F value Pr(>F)
## Condition 1 1154.6 1154.6 48.609 3.29e-07 ***
## Treatment 5 257.5 51.5 2.168 0.0916 .
## Condition:Treatment 5 117.1 23.4 0.986 0.4467
## Residuals 24 570.0 23.8
## ---
## Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1# testing homogeneity of variances
plot(proline.wk8.aov, 1)
leveneTest(Proline ~ Condition*Treatment, data = proline.wk8) # p > 0.05 means no violation of homogeneity of varaince# normality test
plot(proline.wk8.aov, 2)
proline.wk8.residuals <- residuals(object = proline.wk8.aov) # p > 0.05 means no violation of homogeneity of varaince
shapiro.test(x = proline.wk8.residuals)##
## Shapiro-Wilk normality test
##
## data: proline.wk8.residuals
## W = 0.98609, p-value = 0.922proline.boxplot <- ggplot(data = clusia.proline, aes(x = Treatment, y = Proline, fill = Condition)) + geom_boxplot() + geom_point(aes(fill = Condition), size = 2, shape = 21, position = position_jitterdodge()) + facet_grid(. ~ Week)
proline.boxplot <- proline.boxplot + theme_bw() +scale_fill_manual(values=c("indianred3", "cadetblue3"), breaks=c("Well-watered", "Drought"),labels=c("Well-watered", "Drought")) + theme(panel.grid.major = element_blank(), panel.grid.minor = element_blank()) + theme(axis.title.x = element_blank()) + theme(axis.text.x = element_text(angle = 45, hjust = 1, vjust = 1)) + theme(legend.position = c(0.09, 0.85))
pdf("Proline_Boxplot.pdf")
plot(proline.boxplot)
dev.off()## quartz_off_screen
## 2Water is predominantly the determinant of proline concentration, though not until Week 4. Unlike malic acid and lipid peroxidation, there is a slight influence of microbial community on proline in Week 6 and Week 8 (p-value < 0.1), though Tukeys HSD shows that these differences are the result of differences between well-watered communities and droughted communities. Therefore, the significant interaction term is not an indication that plants containing each constructed communities respond differently within the drought treatment. Well-watered plants have significantly lower amounts of proline than drought plants at Week 4 (p-value < 0.05), with the gap between the two widening over time. This is consistent with proline being a stress metabolite that increases in response to drought stress.
Osmolality
osmo <- read.csv("~/Documents/LabStats/Clusia/Osmolality.csv", header = TRUE, stringsAsFactors = TRUE)
osmo <- osmo %>%
arrange(Week, Condition, Treatment)Controls
# Week 0
wk0.cont <- osmo %>%
filter(Treatment == "Control" | Treatment == "Control Mock") %>%
filter(Week == "Wk_0")
wk0.cont.aov <- aov(Osmolality ~ Condition * Treatment, data = wk0.cont)
summary(wk0.cont.aov) # no significant differences between control mock and control## Df Sum Sq Mean Sq F value Pr(>F)
## Condition 1 107 107 0.024 0.880
## Treatment 1 1574 1574 0.358 0.569
## Condition:Treatment 1 5541 5541 1.259 0.299
## Residuals 7 30811 4402# Week 2
wk2.cont <- osmo %>%
filter(Treatment == "Control" | Treatment == "Control Mock") %>%
filter(Week == "Wk_2")
wk2.cont.aov <- aov(Osmolality ~ Condition * Treatment, data = wk2.cont)
summary(wk2.cont.aov) # no significant differences between control mock and control## Df Sum Sq Mean Sq F value Pr(>F)
## Condition 1 62845 62845 44.355 0.000288 ***
## Treatment 1 1229 1229 0.867 0.382707
## Condition:Treatment 1 0 0 0.000 1.000000
## Residuals 7 9918 1417
## ---
## Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1# Week 4
wk4.cont <- osmo %>%
filter(Treatment == "Control" | Treatment == "Control Mock") %>%
filter(Week == "Wk_4")
wk4.cont.aov <- aov(Osmolality ~ Condition * Treatment, data = wk4.cont)
summary(wk4.cont.aov) # no significant differences between control mock and control## Df Sum Sq Mean Sq F value Pr(>F)
## Condition 1 85957 85957 19.207 0.00466 **
## Treatment 1 1562 1562 0.349 0.57617
## Condition:Treatment 1 7370 7370 1.647 0.24672
## Residuals 6 26852 4475
## ---
## Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1# Week 6
wk6.cont <- osmo %>%
filter(Treatment == "Control" | Treatment == "Control Mock") %>%
filter(Week == "Wk_6")
wk6.cont.aov <- aov(Osmolality ~ Condition * Treatment, data = wk6.cont)
summary(wk6.cont.aov) # no significant differences between control mock and control## Df Sum Sq Mean Sq F value Pr(>F)
## Condition 1 15110 15110 6.862 0.0344 *
## Treatment 1 3983 3983 1.809 0.2206
## Condition:Treatment 1 1452 1452 0.659 0.4435
## Residuals 7 15413 2202
## ---
## Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1# Week 8
wk8.cont <- osmo %>%
filter(Treatment == "Control" | Treatment == "Control Mock") %>%
filter(Week == "Wk_8")
wk8.cont.aov <- aov(Osmolality ~ Condition * Treatment, data = wk8.cont)
summary(wk8.cont.aov) # no significant differences between control mock and control## Df Sum Sq Mean Sq F value Pr(>F)
## Condition 1 8646 8646 1.371 0.307
## Treatment 1 15400 15400 2.442 0.193
## Condition:Treatment 1 5050 5050 0.801 0.421
## Residuals 4 25228 6307# removing Control Mocks
osmo <- osmo %>%
filter(Treatment != "Control Mock")Mean, SD, SE
osmo$Week[osmo$Week == "WK_8"] <- "Wk_8" # fixing spelling error
osmo.sum <- osmo %>%
group_by(Week, Condition, Treatment) %>%
summarise(n = n(),
mean = mean(Osmolality, na.rm = TRUE),
sd = sd(Osmolality, na.rm = TRUE),
se = sd(Osmolality, na.rm = TRUE)/sqrt(n()))
View(osmo.sum)
osmo.errobars <- ggplot(data = osmo.sum, aes(x = Treatment, y = mean, fill = Condition)) + geom_bar(stat = "identity", color = "black", position = position_dodge(0.9)) + geom_errorbar(aes(ymin = mean-sd, ymax = mean+sd), width = 0.2, position = position_dodge(0.9)) + facet_grid(. ~ Week)
osmo.errobars <- osmo.errobars + theme_bw() + scale_fill_manual(values=c("indianred3", "cadetblue3"), breaks=c("Well-watered", "Drought"),labels=c("Well-watered", "Drought")) + theme(panel.grid.major = element_blank(), panel.grid.minor = element_blank()) + theme(axis.title.x = element_blank()) + theme(axis.text.x = element_text(angle = 45, hjust = 1, vjust = 1)) + ylab(expression(Mean~Osmolality))
osmo.errobars## Warning: Removed 1 rows containing missing values (geom_errorbar).
### ANOVAs #### Week 0
# Week 0
osmo.wk0 <- osmo %>%
filter(Week == "Wk_0")
# density histogram
ggplot(data = osmo.wk0, aes(x = Osmolality)) + geom_histogram(binwidth = 30, aes(y =..density..), colour = "black", fill = "white") + geom_density(alpha = 0.2, fill = "#FF6666") 
# by water treatment
osmo.wk0.mu <- osmo.wk0 %>%
group_by(Condition) %>%
summarise(mean = mean(Osmolality, na.rm = TRUE))
ggplot(data = osmo.wk0, aes(x = Osmolality, fill = Condition)) + geom_histogram(binwidth = 30, position = "identity", alpha = 0.5) + geom_vline(data = osmo.wk0.mu, aes(xintercept=mean, color=Condition), linetype="dashed") + scale_color_manual(values=c("indianred3", "cadetblue3")) + theme_classic() + xlab("Osmolality") + ylab("Count") + theme(legend.position = c(0.9, 0.9))
# ANOVA
osmo.wk0.aov <- aov(Osmolality ~ Condition * Treatment, data = osmo.wk0)
summary(osmo.wk0.aov)## Df Sum Sq Mean Sq F value Pr(>F)
## Condition 1 6 6 0.002 0.969
## Treatment 4 7541 1885 0.480 0.750
## Condition:Treatment 4 8850 2212 0.563 0.692
## Residuals 21 82545 3931# testing homogeneity of variances
plot(osmo.wk0.aov, 1)
leveneTest(Osmolality ~ Condition*Treatment, data = osmo.wk0) # p > 0.05 means no violation of homogeneity of varaince# normality test
plot(osmo.wk0.aov, 2)
osmo.wk0.residuals <- residuals(object = osmo.wk0.aov) # p > 0.05 means no violation of homogeneity of varaince
shapiro.test(x = osmo.wk0.residuals)##
## Shapiro-Wilk normality test
##
## data: osmo.wk0.residuals
## W = 0.9629, p-value = 0.3475Week 2
# Week 2
wk2 <- osmo %>%
filter(Week == "Wk_2")
# ANOVA
wk2.aov <- aov(Osmolality ~ Condition * Treatment, data = wk2)
summary(wk2.aov)## Df Sum Sq Mean Sq F value Pr(>F)
## Condition 1 116439 116439 37.792 5.25e-06 ***
## Treatment 4 19690 4923 1.598 0.21380
## Condition:Treatment 4 62910 15728 5.105 0.00533 **
## Residuals 20 61621 3081
## ---
## Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1Week 4
# Week 4
osmo.wk4 <- osmo %>%
filter(Week == "Wk_4")
# density histogram
ggplot(data = osmo.wk4, aes(x = Osmolality)) + geom_histogram(binwidth = 52, aes(y =..density..), colour = "black", fill = "white") + geom_density(alpha = 0.2, fill = "#FF6666") 
# by water treatment
osmo.wk4.mu <- osmo.wk4 %>%
group_by(Condition) %>%
summarise(mean = mean(Osmolality, na.rm = TRUE))
ggplot(data = osmo.wk4, aes(x = Osmolality, fill = Condition)) + geom_histogram(binwidth = 52, position = "identity", alpha = 0.5) + geom_vline(data = osmo.wk4.mu, aes(xintercept=mean, color=Condition), linetype="dashed") + scale_color_manual(values=c("indianred3", "cadetblue3")) + theme_classic() + xlab("Osmolality") + ylab("Count") + theme(legend.position = c(0.9, 0.9))
osmo.wk4.aov <- aov(Osmolality ~ Condition * Treatment, data = osmo.wk4)
summary(osmo.wk4.aov) # significant drought, microbial, and interaction term## Df Sum Sq Mean Sq F value Pr(>F)
## Condition 1 214760 214760 123.827 6.09e-09 ***
## Treatment 4 42077 10519 6.065 0.00362 **
## Condition:Treatment 4 17167 4292 2.475 0.08611 .
## Residuals 16 27750 1734
## ---
## Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1TukeyHSD(osmo.wk4.aov)## Tukey multiple comparisons of means
## 95% family-wise confidence level
##
## Fit: aov(formula = Osmolality ~ Condition * Treatment, data = osmo.wk4)
##
## $Condition
## diff lwr upr p adj
## Well-watered-Drought 181.7692 147.141 216.3974 0
##
## $Treatment
## diff lwr
## Control-All Isolates -35.123077 -112.381940
## Drought Isolates-All Isolates 96.205128 5.986213
## Nonfunctional Isolates-All Isolates -36.500000 -110.163436
## Phosphorus Isolates-All Isolates -8.000000 -81.663436
## Drought Isolates-Control 131.328205 38.150510
## Nonfunctional Isolates-Control -1.376923 -78.635787
## Phosphorus Isolates-Control 27.123077 -50.135787
## Nonfunctional Isolates-Drought Isolates -132.705128 -222.924044
## Phosphorus Isolates-Drought Isolates -104.205128 -194.424044
## Phosphorus Isolates-Nonfunctional Isolates 28.500000 -45.163436
## upr p adj
## Control-All Isolates 42.13579 0.6405864
## Drought Isolates-All Isolates 186.42404 0.0337916
## Nonfunctional Isolates-All Isolates 37.16344 0.5661953
## Phosphorus Isolates-All Isolates 65.66344 0.9970701
## Drought Isolates-Control 224.50590 0.0041556
## Nonfunctional Isolates-Control 75.88194 0.9999977
## Phosphorus Isolates-Control 104.38194 0.8161977
## Nonfunctional Isolates-Drought Isolates -42.48621 0.0028489
## Phosphorus Isolates-Drought Isolates -13.98621 0.0198071
## Phosphorus Isolates-Nonfunctional Isolates 102.16344 0.7594089
##
## $`Condition:Treatment`
## diff
## Well-watered:All Isolates-Drought:All Isolates 193.000000
## Drought:Control-Drought:All Isolates -10.333333
## Well-watered:Control-Drought:All Isolates 123.500000
## Drought:Drought Isolates-Drought:All Isolates 153.000000
## Well-watered:Drought Isolates-Drought:All Isolates 258.000000
## Drought:Nonfunctional Isolates-Drought:All Isolates -5.333333
## Well-watered:Nonfunctional Isolates-Drought:All Isolates 125.333333
## Drought:Phosphorus Isolates-Drought:All Isolates -36.666667
## Well-watered:Phosphorus Isolates-Drought:All Isolates 213.666667
## Drought:Control-Well-watered:All Isolates -203.333333
## Well-watered:Control-Well-watered:All Isolates -69.500000
## Drought:Drought Isolates-Well-watered:All Isolates -40.000000
## Well-watered:Drought Isolates-Well-watered:All Isolates 65.000000
## Drought:Nonfunctional Isolates-Well-watered:All Isolates -198.333333
## Well-watered:Nonfunctional Isolates-Well-watered:All Isolates -67.666667
## Drought:Phosphorus Isolates-Well-watered:All Isolates -229.666667
## Well-watered:Phosphorus Isolates-Well-watered:All Isolates 20.666667
## Well-watered:Control-Drought:Control 133.833333
## Drought:Drought Isolates-Drought:Control 163.333333
## Well-watered:Drought Isolates-Drought:Control 268.333333
## Drought:Nonfunctional Isolates-Drought:Control 5.000000
## Well-watered:Nonfunctional Isolates-Drought:Control 135.666667
## Drought:Phosphorus Isolates-Drought:Control -26.333333
## Well-watered:Phosphorus Isolates-Drought:Control 224.000000
## Drought:Drought Isolates-Well-watered:Control 29.500000
## Well-watered:Drought Isolates-Well-watered:Control 134.500000
## Drought:Nonfunctional Isolates-Well-watered:Control -128.833333
## Well-watered:Nonfunctional Isolates-Well-watered:Control 1.833333
## Drought:Phosphorus Isolates-Well-watered:Control -160.166667
## Well-watered:Phosphorus Isolates-Well-watered:Control 90.166667
## Well-watered:Drought Isolates-Drought:Drought Isolates 105.000000
## Drought:Nonfunctional Isolates-Drought:Drought Isolates -158.333333
## Well-watered:Nonfunctional Isolates-Drought:Drought Isolates -27.666667
## Drought:Phosphorus Isolates-Drought:Drought Isolates -189.666667
## Well-watered:Phosphorus Isolates-Drought:Drought Isolates 60.666667
## Drought:Nonfunctional Isolates-Well-watered:Drought Isolates -263.333333
## Well-watered:Nonfunctional Isolates-Well-watered:Drought Isolates -132.666667
## Drought:Phosphorus Isolates-Well-watered:Drought Isolates -294.666667
## Well-watered:Phosphorus Isolates-Well-watered:Drought Isolates -44.333333
## Well-watered:Nonfunctional Isolates-Drought:Nonfunctional Isolates 130.666667
## Drought:Phosphorus Isolates-Drought:Nonfunctional Isolates -31.333333
## Well-watered:Phosphorus Isolates-Drought:Nonfunctional Isolates 219.000000
## Drought:Phosphorus Isolates-Well-watered:Nonfunctional Isolates -162.000000
## Well-watered:Phosphorus Isolates-Well-watered:Nonfunctional Isolates 88.333333
## Well-watered:Phosphorus Isolates-Drought:Phosphorus Isolates 250.333333
## lwr
## Well-watered:All Isolates-Drought:All Isolates 69.177608
## Drought:Control-Drought:All Isolates -134.155725
## Well-watered:Control-Drought:All Isolates -14.937642
## Drought:Drought Isolates-Drought:All Isolates -22.111306
## Well-watered:Drought Isolates-Drought:All Isolates 119.562358
## Drought:Nonfunctional Isolates-Drought:All Isolates -129.155725
## Well-watered:Nonfunctional Isolates-Drought:All Isolates 1.510942
## Drought:Phosphorus Isolates-Drought:All Isolates -160.489058
## Well-watered:Phosphorus Isolates-Drought:All Isolates 89.844275
## Drought:Control-Well-watered:All Isolates -327.155725
## Well-watered:Control-Well-watered:All Isolates -207.937642
## Drought:Drought Isolates-Well-watered:All Isolates -215.111306
## Well-watered:Drought Isolates-Well-watered:All Isolates -73.437642
## Drought:Nonfunctional Isolates-Well-watered:All Isolates -322.155725
## Well-watered:Nonfunctional Isolates-Well-watered:All Isolates -191.489058
## Drought:Phosphorus Isolates-Well-watered:All Isolates -353.489058
## Well-watered:Phosphorus Isolates-Well-watered:All Isolates -103.155725
## Well-watered:Control-Drought:Control -4.604309
## Drought:Drought Isolates-Drought:Control -11.777972
## Well-watered:Drought Isolates-Drought:Control 129.895691
## Drought:Nonfunctional Isolates-Drought:Control -118.822392
## Well-watered:Nonfunctional Isolates-Drought:Control 11.844275
## Drought:Phosphorus Isolates-Drought:Control -150.155725
## Well-watered:Phosphorus Isolates-Drought:Control 100.177608
## Drought:Drought Isolates-Well-watered:Control -156.233587
## Well-watered:Drought Isolates-Well-watered:Control -17.150839
## Drought:Nonfunctional Isolates-Well-watered:Control -267.270976
## Well-watered:Nonfunctional Isolates-Well-watered:Control -136.604309
## Drought:Phosphorus Isolates-Well-watered:Control -298.604309
## Well-watered:Phosphorus Isolates-Well-watered:Control -48.270976
## Well-watered:Drought Isolates-Drought:Drought Isolates -80.733587
## Drought:Nonfunctional Isolates-Drought:Drought Isolates -333.444639
## Well-watered:Nonfunctional Isolates-Drought:Drought Isolates -202.777972
## Drought:Phosphorus Isolates-Drought:Drought Isolates -364.777972
## Well-watered:Phosphorus Isolates-Drought:Drought Isolates -114.444639
## Drought:Nonfunctional Isolates-Well-watered:Drought Isolates -401.770976
## Well-watered:Nonfunctional Isolates-Well-watered:Drought Isolates -271.104309
## Drought:Phosphorus Isolates-Well-watered:Drought Isolates -433.104309
## Well-watered:Phosphorus Isolates-Well-watered:Drought Isolates -182.770976
## Well-watered:Nonfunctional Isolates-Drought:Nonfunctional Isolates 6.844275
## Drought:Phosphorus Isolates-Drought:Nonfunctional Isolates -155.155725
## Well-watered:Phosphorus Isolates-Drought:Nonfunctional Isolates 95.177608
## Drought:Phosphorus Isolates-Well-watered:Nonfunctional Isolates -285.822392
## Well-watered:Phosphorus Isolates-Well-watered:Nonfunctional Isolates -35.489058
## Well-watered:Phosphorus Isolates-Drought:Phosphorus Isolates 126.510942
## upr
## Well-watered:All Isolates-Drought:All Isolates 316.822392
## Drought:Control-Drought:All Isolates 113.489058
## Well-watered:Control-Drought:All Isolates 261.937642
## Drought:Drought Isolates-Drought:All Isolates 328.111306
## Well-watered:Drought Isolates-Drought:All Isolates 396.437642
## Drought:Nonfunctional Isolates-Drought:All Isolates 118.489058
## Well-watered:Nonfunctional Isolates-Drought:All Isolates 249.155725
## Drought:Phosphorus Isolates-Drought:All Isolates 87.155725
## Well-watered:Phosphorus Isolates-Drought:All Isolates 337.489058
## Drought:Control-Well-watered:All Isolates -79.510942
## Well-watered:Control-Well-watered:All Isolates 68.937642
## Drought:Drought Isolates-Well-watered:All Isolates 135.111306
## Well-watered:Drought Isolates-Well-watered:All Isolates 203.437642
## Drought:Nonfunctional Isolates-Well-watered:All Isolates -74.510942
## Well-watered:Nonfunctional Isolates-Well-watered:All Isolates 56.155725
## Drought:Phosphorus Isolates-Well-watered:All Isolates -105.844275
## Well-watered:Phosphorus Isolates-Well-watered:All Isolates 144.489058
## Well-watered:Control-Drought:Control 272.270976
## Drought:Drought Isolates-Drought:Control 338.444639
## Well-watered:Drought Isolates-Drought:Control 406.770976
## Drought:Nonfunctional Isolates-Drought:Control 128.822392
## Well-watered:Nonfunctional Isolates-Drought:Control 259.489058
## Drought:Phosphorus Isolates-Drought:Control 97.489058
## Well-watered:Phosphorus Isolates-Drought:Control 347.822392
## Drought:Drought Isolates-Well-watered:Control 215.233587
## Well-watered:Drought Isolates-Well-watered:Control 286.150839
## Drought:Nonfunctional Isolates-Well-watered:Control 9.604309
## Well-watered:Nonfunctional Isolates-Well-watered:Control 140.270976
## Drought:Phosphorus Isolates-Well-watered:Control -21.729024
## Well-watered:Phosphorus Isolates-Well-watered:Control 228.604309
## Well-watered:Drought Isolates-Drought:Drought Isolates 290.733587
## Drought:Nonfunctional Isolates-Drought:Drought Isolates 16.777972
## Well-watered:Nonfunctional Isolates-Drought:Drought Isolates 147.444639
## Drought:Phosphorus Isolates-Drought:Drought Isolates -14.555361
## Well-watered:Phosphorus Isolates-Drought:Drought Isolates 235.777972
## Drought:Nonfunctional Isolates-Well-watered:Drought Isolates -124.895691
## Well-watered:Nonfunctional Isolates-Well-watered:Drought Isolates 5.770976
## Drought:Phosphorus Isolates-Well-watered:Drought Isolates -156.229024
## Well-watered:Phosphorus Isolates-Well-watered:Drought Isolates 94.104309
## Well-watered:Nonfunctional Isolates-Drought:Nonfunctional Isolates 254.489058
## Drought:Phosphorus Isolates-Drought:Nonfunctional Isolates 92.489058
## Well-watered:Phosphorus Isolates-Drought:Nonfunctional Isolates 342.822392
## Drought:Phosphorus Isolates-Well-watered:Nonfunctional Isolates -38.177608
## Well-watered:Phosphorus Isolates-Well-watered:Nonfunctional Isolates 212.155725
## Well-watered:Phosphorus Isolates-Drought:Phosphorus Isolates 374.155725
## p adj
## Well-watered:All Isolates-Drought:All Isolates 0.0010483
## Drought:Control-Drought:All Isolates 0.9999992
## Well-watered:Control-Drought:All Isolates 0.1017466
## Drought:Drought Isolates-Drought:All Isolates 0.1143441
## Well-watered:Drought Isolates-Drought:All Isolates 0.0001417
## Drought:Nonfunctional Isolates-Drought:All Isolates 1.0000000
## Well-watered:Nonfunctional Isolates-Drought:All Isolates 0.0460426
## Drought:Phosphorus Isolates-Drought:All Isolates 0.9802717
## Well-watered:Phosphorus Isolates-Drought:All Isolates 0.0003449
## Drought:Control-Well-watered:All Isolates 0.0005984
## Well-watered:Control-Well-watered:All Isolates 0.7110510
## Drought:Drought Isolates-Well-watered:All Isolates 0.9966917
## Well-watered:Drought Isolates-Well-watered:All Isolates 0.7759432
## Drought:Nonfunctional Isolates-Well-watered:All Isolates 0.0007840
## Well-watered:Nonfunctional Isolates-Well-watered:All Isolates 0.6164630
## Drought:Phosphorus Isolates-Well-watered:All Isolates 0.0001499
## Well-watered:Phosphorus Isolates-Well-watered:All Isolates 0.9997052
## Well-watered:Control-Drought:Control 0.0624818
## Drought:Drought Isolates-Drought:Control 0.0782095
## Well-watered:Drought Isolates-Drought:Control 0.0000887
## Drought:Nonfunctional Isolates-Drought:Control 1.0000000
## Well-watered:Nonfunctional Isolates-Drought:Control 0.0259965
## Drought:Phosphorus Isolates-Drought:Control 0.9980522
## Well-watered:Phosphorus Isolates-Drought:Control 0.0002008
## Drought:Drought Isolates-Well-watered:Control 0.9998025
## Well-watered:Drought Isolates-Well-watered:Control 0.1051756
## Drought:Nonfunctional Isolates-Well-watered:Control 0.0793003
## Well-watered:Nonfunctional Isolates-Well-watered:Control 1.0000000
## Drought:Phosphorus Isolates-Well-watered:Control 0.0169822
## Well-watered:Phosphorus Isolates-Well-watered:Control 0.3994380
## Well-watered:Drought Isolates-Drought:Drought Isolates 0.5757429
## Drought:Nonfunctional Isolates-Drought:Drought Isolates 0.0941504
## Well-watered:Nonfunctional Isolates-Drought:Drought Isolates 0.9998108
## Drought:Phosphorus Isolates-Drought:Drought Isolates 0.0283471
## Well-watered:Phosphorus Isolates-Drought:Drought Isolates 0.9490213
## Drought:Nonfunctional Isolates-Well-watered:Drought Isolates 0.0001112
## Well-watered:Nonfunctional Isolates-Well-watered:Drought Isolates 0.0660794
## Drought:Phosphorus Isolates-Well-watered:Drought Isolates 0.0000280
## Well-watered:Phosphorus Isolates-Well-watered:Drought Isolates 0.9678755
## Well-watered:Nonfunctional Isolates-Drought:Nonfunctional Isolates 0.0343302
## Drought:Phosphorus Isolates-Drought:Nonfunctional Isolates 0.9931191
## Well-watered:Phosphorus Isolates-Drought:Nonfunctional Isolates 0.0002605
## Drought:Phosphorus Isolates-Well-watered:Nonfunctional Isolates 0.0058933
## Well-watered:Phosphorus Isolates-Well-watered:Nonfunctional Isolates 0.2925005
## Well-watered:Phosphorus Isolates-Drought:Phosphorus Isolates 0.0000531# ANOVA
osmo.wk4.aov <- aov(Osmolality ~ Condition * Treatment, data = osmo.wk4)
summary(osmo.wk4.aov) # significant drought, microbial, and interaction term## Df Sum Sq Mean Sq F value Pr(>F)
## Condition 1 214760 214760 123.827 6.09e-09 ***
## Treatment 4 42077 10519 6.065 0.00362 **
## Condition:Treatment 4 17167 4292 2.475 0.08611 .
## Residuals 16 27750 1734
## ---
## Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1TukeyHSD(osmo.wk4.aov)## Tukey multiple comparisons of means
## 95% family-wise confidence level
##
## Fit: aov(formula = Osmolality ~ Condition * Treatment, data = osmo.wk4)
##
## $Condition
## diff lwr upr p adj
## Well-watered-Drought 181.7692 147.141 216.3974 0
##
## $Treatment
## diff lwr
## Control-All Isolates -35.123077 -112.381940
## Drought Isolates-All Isolates 96.205128 5.986213
## Nonfunctional Isolates-All Isolates -36.500000 -110.163436
## Phosphorus Isolates-All Isolates -8.000000 -81.663436
## Drought Isolates-Control 131.328205 38.150510
## Nonfunctional Isolates-Control -1.376923 -78.635787
## Phosphorus Isolates-Control 27.123077 -50.135787
## Nonfunctional Isolates-Drought Isolates -132.705128 -222.924044
## Phosphorus Isolates-Drought Isolates -104.205128 -194.424044
## Phosphorus Isolates-Nonfunctional Isolates 28.500000 -45.163436
## upr p adj
## Control-All Isolates 42.13579 0.6405864
## Drought Isolates-All Isolates 186.42404 0.0337916
## Nonfunctional Isolates-All Isolates 37.16344 0.5661953
## Phosphorus Isolates-All Isolates 65.66344 0.9970701
## Drought Isolates-Control 224.50590 0.0041556
## Nonfunctional Isolates-Control 75.88194 0.9999977
## Phosphorus Isolates-Control 104.38194 0.8161977
## Nonfunctional Isolates-Drought Isolates -42.48621 0.0028489
## Phosphorus Isolates-Drought Isolates -13.98621 0.0198071
## Phosphorus Isolates-Nonfunctional Isolates 102.16344 0.7594089
##
## $`Condition:Treatment`
## diff
## Well-watered:All Isolates-Drought:All Isolates 193.000000
## Drought:Control-Drought:All Isolates -10.333333
## Well-watered:Control-Drought:All Isolates 123.500000
## Drought:Drought Isolates-Drought:All Isolates 153.000000
## Well-watered:Drought Isolates-Drought:All Isolates 258.000000
## Drought:Nonfunctional Isolates-Drought:All Isolates -5.333333
## Well-watered:Nonfunctional Isolates-Drought:All Isolates 125.333333
## Drought:Phosphorus Isolates-Drought:All Isolates -36.666667
## Well-watered:Phosphorus Isolates-Drought:All Isolates 213.666667
## Drought:Control-Well-watered:All Isolates -203.333333
## Well-watered:Control-Well-watered:All Isolates -69.500000
## Drought:Drought Isolates-Well-watered:All Isolates -40.000000
## Well-watered:Drought Isolates-Well-watered:All Isolates 65.000000
## Drought:Nonfunctional Isolates-Well-watered:All Isolates -198.333333
## Well-watered:Nonfunctional Isolates-Well-watered:All Isolates -67.666667
## Drought:Phosphorus Isolates-Well-watered:All Isolates -229.666667
## Well-watered:Phosphorus Isolates-Well-watered:All Isolates 20.666667
## Well-watered:Control-Drought:Control 133.833333
## Drought:Drought Isolates-Drought:Control 163.333333
## Well-watered:Drought Isolates-Drought:Control 268.333333
## Drought:Nonfunctional Isolates-Drought:Control 5.000000
## Well-watered:Nonfunctional Isolates-Drought:Control 135.666667
## Drought:Phosphorus Isolates-Drought:Control -26.333333
## Well-watered:Phosphorus Isolates-Drought:Control 224.000000
## Drought:Drought Isolates-Well-watered:Control 29.500000
## Well-watered:Drought Isolates-Well-watered:Control 134.500000
## Drought:Nonfunctional Isolates-Well-watered:Control -128.833333
## Well-watered:Nonfunctional Isolates-Well-watered:Control 1.833333
## Drought:Phosphorus Isolates-Well-watered:Control -160.166667
## Well-watered:Phosphorus Isolates-Well-watered:Control 90.166667
## Well-watered:Drought Isolates-Drought:Drought Isolates 105.000000
## Drought:Nonfunctional Isolates-Drought:Drought Isolates -158.333333
## Well-watered:Nonfunctional Isolates-Drought:Drought Isolates -27.666667
## Drought:Phosphorus Isolates-Drought:Drought Isolates -189.666667
## Well-watered:Phosphorus Isolates-Drought:Drought Isolates 60.666667
## Drought:Nonfunctional Isolates-Well-watered:Drought Isolates -263.333333
## Well-watered:Nonfunctional Isolates-Well-watered:Drought Isolates -132.666667
## Drought:Phosphorus Isolates-Well-watered:Drought Isolates -294.666667
## Well-watered:Phosphorus Isolates-Well-watered:Drought Isolates -44.333333
## Well-watered:Nonfunctional Isolates-Drought:Nonfunctional Isolates 130.666667
## Drought:Phosphorus Isolates-Drought:Nonfunctional Isolates -31.333333
## Well-watered:Phosphorus Isolates-Drought:Nonfunctional Isolates 219.000000
## Drought:Phosphorus Isolates-Well-watered:Nonfunctional Isolates -162.000000
## Well-watered:Phosphorus Isolates-Well-watered:Nonfunctional Isolates 88.333333
## Well-watered:Phosphorus Isolates-Drought:Phosphorus Isolates 250.333333
## lwr
## Well-watered:All Isolates-Drought:All Isolates 69.177608
## Drought:Control-Drought:All Isolates -134.155725
## Well-watered:Control-Drought:All Isolates -14.937642
## Drought:Drought Isolates-Drought:All Isolates -22.111306
## Well-watered:Drought Isolates-Drought:All Isolates 119.562358
## Drought:Nonfunctional Isolates-Drought:All Isolates -129.155725
## Well-watered:Nonfunctional Isolates-Drought:All Isolates 1.510942
## Drought:Phosphorus Isolates-Drought:All Isolates -160.489058
## Well-watered:Phosphorus Isolates-Drought:All Isolates 89.844275
## Drought:Control-Well-watered:All Isolates -327.155725
## Well-watered:Control-Well-watered:All Isolates -207.937642
## Drought:Drought Isolates-Well-watered:All Isolates -215.111306
## Well-watered:Drought Isolates-Well-watered:All Isolates -73.437642
## Drought:Nonfunctional Isolates-Well-watered:All Isolates -322.155725
## Well-watered:Nonfunctional Isolates-Well-watered:All Isolates -191.489058
## Drought:Phosphorus Isolates-Well-watered:All Isolates -353.489058
## Well-watered:Phosphorus Isolates-Well-watered:All Isolates -103.155725
## Well-watered:Control-Drought:Control -4.604309
## Drought:Drought Isolates-Drought:Control -11.777972
## Well-watered:Drought Isolates-Drought:Control 129.895691
## Drought:Nonfunctional Isolates-Drought:Control -118.822392
## Well-watered:Nonfunctional Isolates-Drought:Control 11.844275
## Drought:Phosphorus Isolates-Drought:Control -150.155725
## Well-watered:Phosphorus Isolates-Drought:Control 100.177608
## Drought:Drought Isolates-Well-watered:Control -156.233587
## Well-watered:Drought Isolates-Well-watered:Control -17.150839
## Drought:Nonfunctional Isolates-Well-watered:Control -267.270976
## Well-watered:Nonfunctional Isolates-Well-watered:Control -136.604309
## Drought:Phosphorus Isolates-Well-watered:Control -298.604309
## Well-watered:Phosphorus Isolates-Well-watered:Control -48.270976
## Well-watered:Drought Isolates-Drought:Drought Isolates -80.733587
## Drought:Nonfunctional Isolates-Drought:Drought Isolates -333.444639
## Well-watered:Nonfunctional Isolates-Drought:Drought Isolates -202.777972
## Drought:Phosphorus Isolates-Drought:Drought Isolates -364.777972
## Well-watered:Phosphorus Isolates-Drought:Drought Isolates -114.444639
## Drought:Nonfunctional Isolates-Well-watered:Drought Isolates -401.770976
## Well-watered:Nonfunctional Isolates-Well-watered:Drought Isolates -271.104309
## Drought:Phosphorus Isolates-Well-watered:Drought Isolates -433.104309
## Well-watered:Phosphorus Isolates-Well-watered:Drought Isolates -182.770976
## Well-watered:Nonfunctional Isolates-Drought:Nonfunctional Isolates 6.844275
## Drought:Phosphorus Isolates-Drought:Nonfunctional Isolates -155.155725
## Well-watered:Phosphorus Isolates-Drought:Nonfunctional Isolates 95.177608
## Drought:Phosphorus Isolates-Well-watered:Nonfunctional Isolates -285.822392
## Well-watered:Phosphorus Isolates-Well-watered:Nonfunctional Isolates -35.489058
## Well-watered:Phosphorus Isolates-Drought:Phosphorus Isolates 126.510942
## upr
## Well-watered:All Isolates-Drought:All Isolates 316.822392
## Drought:Control-Drought:All Isolates 113.489058
## Well-watered:Control-Drought:All Isolates 261.937642
## Drought:Drought Isolates-Drought:All Isolates 328.111306
## Well-watered:Drought Isolates-Drought:All Isolates 396.437642
## Drought:Nonfunctional Isolates-Drought:All Isolates 118.489058
## Well-watered:Nonfunctional Isolates-Drought:All Isolates 249.155725
## Drought:Phosphorus Isolates-Drought:All Isolates 87.155725
## Well-watered:Phosphorus Isolates-Drought:All Isolates 337.489058
## Drought:Control-Well-watered:All Isolates -79.510942
## Well-watered:Control-Well-watered:All Isolates 68.937642
## Drought:Drought Isolates-Well-watered:All Isolates 135.111306
## Well-watered:Drought Isolates-Well-watered:All Isolates 203.437642
## Drought:Nonfunctional Isolates-Well-watered:All Isolates -74.510942
## Well-watered:Nonfunctional Isolates-Well-watered:All Isolates 56.155725
## Drought:Phosphorus Isolates-Well-watered:All Isolates -105.844275
## Well-watered:Phosphorus Isolates-Well-watered:All Isolates 144.489058
## Well-watered:Control-Drought:Control 272.270976
## Drought:Drought Isolates-Drought:Control 338.444639
## Well-watered:Drought Isolates-Drought:Control 406.770976
## Drought:Nonfunctional Isolates-Drought:Control 128.822392
## Well-watered:Nonfunctional Isolates-Drought:Control 259.489058
## Drought:Phosphorus Isolates-Drought:Control 97.489058
## Well-watered:Phosphorus Isolates-Drought:Control 347.822392
## Drought:Drought Isolates-Well-watered:Control 215.233587
## Well-watered:Drought Isolates-Well-watered:Control 286.150839
## Drought:Nonfunctional Isolates-Well-watered:Control 9.604309
## Well-watered:Nonfunctional Isolates-Well-watered:Control 140.270976
## Drought:Phosphorus Isolates-Well-watered:Control -21.729024
## Well-watered:Phosphorus Isolates-Well-watered:Control 228.604309
## Well-watered:Drought Isolates-Drought:Drought Isolates 290.733587
## Drought:Nonfunctional Isolates-Drought:Drought Isolates 16.777972
## Well-watered:Nonfunctional Isolates-Drought:Drought Isolates 147.444639
## Drought:Phosphorus Isolates-Drought:Drought Isolates -14.555361
## Well-watered:Phosphorus Isolates-Drought:Drought Isolates 235.777972
## Drought:Nonfunctional Isolates-Well-watered:Drought Isolates -124.895691
## Well-watered:Nonfunctional Isolates-Well-watered:Drought Isolates 5.770976
## Drought:Phosphorus Isolates-Well-watered:Drought Isolates -156.229024
## Well-watered:Phosphorus Isolates-Well-watered:Drought Isolates 94.104309
## Well-watered:Nonfunctional Isolates-Drought:Nonfunctional Isolates 254.489058
## Drought:Phosphorus Isolates-Drought:Nonfunctional Isolates 92.489058
## Well-watered:Phosphorus Isolates-Drought:Nonfunctional Isolates 342.822392
## Drought:Phosphorus Isolates-Well-watered:Nonfunctional Isolates -38.177608
## Well-watered:Phosphorus Isolates-Well-watered:Nonfunctional Isolates 212.155725
## Well-watered:Phosphorus Isolates-Drought:Phosphorus Isolates 374.155725
## p adj
## Well-watered:All Isolates-Drought:All Isolates 0.0010483
## Drought:Control-Drought:All Isolates 0.9999992
## Well-watered:Control-Drought:All Isolates 0.1017466
## Drought:Drought Isolates-Drought:All Isolates 0.1143441
## Well-watered:Drought Isolates-Drought:All Isolates 0.0001417
## Drought:Nonfunctional Isolates-Drought:All Isolates 1.0000000
## Well-watered:Nonfunctional Isolates-Drought:All Isolates 0.0460426
## Drought:Phosphorus Isolates-Drought:All Isolates 0.9802717
## Well-watered:Phosphorus Isolates-Drought:All Isolates 0.0003449
## Drought:Control-Well-watered:All Isolates 0.0005984
## Well-watered:Control-Well-watered:All Isolates 0.7110510
## Drought:Drought Isolates-Well-watered:All Isolates 0.9966917
## Well-watered:Drought Isolates-Well-watered:All Isolates 0.7759432
## Drought:Nonfunctional Isolates-Well-watered:All Isolates 0.0007840
## Well-watered:Nonfunctional Isolates-Well-watered:All Isolates 0.6164630
## Drought:Phosphorus Isolates-Well-watered:All Isolates 0.0001499
## Well-watered:Phosphorus Isolates-Well-watered:All Isolates 0.9997052
## Well-watered:Control-Drought:Control 0.0624818
## Drought:Drought Isolates-Drought:Control 0.0782095
## Well-watered:Drought Isolates-Drought:Control 0.0000887
## Drought:Nonfunctional Isolates-Drought:Control 1.0000000
## Well-watered:Nonfunctional Isolates-Drought:Control 0.0259965
## Drought:Phosphorus Isolates-Drought:Control 0.9980522
## Well-watered:Phosphorus Isolates-Drought:Control 0.0002008
## Drought:Drought Isolates-Well-watered:Control 0.9998025
## Well-watered:Drought Isolates-Well-watered:Control 0.1051756
## Drought:Nonfunctional Isolates-Well-watered:Control 0.0793003
## Well-watered:Nonfunctional Isolates-Well-watered:Control 1.0000000
## Drought:Phosphorus Isolates-Well-watered:Control 0.0169822
## Well-watered:Phosphorus Isolates-Well-watered:Control 0.3994380
## Well-watered:Drought Isolates-Drought:Drought Isolates 0.5757429
## Drought:Nonfunctional Isolates-Drought:Drought Isolates 0.0941504
## Well-watered:Nonfunctional Isolates-Drought:Drought Isolates 0.9998108
## Drought:Phosphorus Isolates-Drought:Drought Isolates 0.0283471
## Well-watered:Phosphorus Isolates-Drought:Drought Isolates 0.9490213
## Drought:Nonfunctional Isolates-Well-watered:Drought Isolates 0.0001112
## Well-watered:Nonfunctional Isolates-Well-watered:Drought Isolates 0.0660794
## Drought:Phosphorus Isolates-Well-watered:Drought Isolates 0.0000280
## Well-watered:Phosphorus Isolates-Well-watered:Drought Isolates 0.9678755
## Well-watered:Nonfunctional Isolates-Drought:Nonfunctional Isolates 0.0343302
## Drought:Phosphorus Isolates-Drought:Nonfunctional Isolates 0.9931191
## Well-watered:Phosphorus Isolates-Drought:Nonfunctional Isolates 0.0002605
## Drought:Phosphorus Isolates-Well-watered:Nonfunctional Isolates 0.0058933
## Well-watered:Phosphorus Isolates-Well-watered:Nonfunctional Isolates 0.2925005
## Well-watered:Phosphorus Isolates-Drought:Phosphorus Isolates 0.0000531# testing homogeneity of variances
plot(osmo.wk4.aov, 1)
leveneTest(Osmolality ~ Condition*Treatment, data = osmo.wk4)# normality test
plot(osmo.wk4.aov, 2)## Warning: not plotting observations with leverage one:
## 7
osmo.wk4.residuals <- residuals(object = osmo.wk4.aov)
shapiro.test(x = osmo.wk4.residuals)##
## Shapiro-Wilk normality test
##
## data: osmo.wk4.residuals
## W = 0.97679, p-value = 0.7996Week 6
# Week 6
wk6 <- osmo %>%
filter(Week == "Wk_6")
# ANOVA
wk6.aov <- aov(Osmolality ~ Condition * Treatment, data = wk6)
summary(wk6.aov) ## Df Sum Sq Mean Sq F value Pr(>F)
## Condition 1 89653 89653 31.968 1.56e-05 ***
## Treatment 4 8888 2222 0.792 0.544
## Condition:Treatment 4 18070 4518 1.611 0.211
## Residuals 20 56090 2804
## ---
## Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1Week 8
# Week 8
osmo.wk8 <- osmo %>%
filter(Week == "Wk_8")
# density histogram
ggplot(data = osmo.wk8, aes(x = Osmolality)) + geom_histogram(binwidth = 110, aes(y =..density..), colour = "black", fill = "white") + geom_density(alpha = 0.2, fill = "#FF6666") 
# by water treatment
osmo.wk8.mu <- osmo.wk8 %>%
group_by(Condition) %>%
summarise(mean = mean(Osmolality, na.rm = TRUE))
ggplot(data = osmo.wk8, aes(x = Osmolality, fill = Condition)) + geom_histogram(binwidth = 110, position = "identity", alpha = 0.5) + geom_vline(data = osmo.wk8.mu, aes(xintercept=mean, color=Condition), linetype="dashed") + scale_color_manual(values=c("indianred3", "cadetblue3")) + theme_classic() + xlab("Osmolality") + ylab("Count") + theme(legend.position = c(0.9, 0.9))
# ANOVA
osmo.wk8.aov <- aov(Osmolality ~ Condition * Treatment, data = osmo.wk8)
summary(osmo.wk8.aov)## Df Sum Sq Mean Sq F value Pr(>F)
## Condition 1 30958 30958 5.718 0.0258 *
## Treatment 4 123800 30950 5.717 0.0026 **
## Condition:Treatment 4 74910 18727 3.459 0.0245 *
## Residuals 22 119104 5414
## ---
## Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1# testing homogeneity of variances
plot(osmo.wk8.aov, 1)
leveneTest(Osmolality ~ Condition*Treatment, data = osmo.wk8) # p < 0.05 means violation of homogeneity of varaince# normality test
plot(osmo.wk8.aov, 2)
osmo.wk8.residuals <- residuals(object = osmo.wk8.aov) # p > 0.05 means no violation of homogeneity of varaince
shapiro.test(x = osmo.wk8.residuals)##
## Shapiro-Wilk normality test
##
## data: osmo.wk8.residuals
## W = 0.97035, p-value = 0.5091